Antibodies and processes for preparing the same

ABSTRACT

Provided herein are various processes for the improved production of antibody producing organisms, antibody producing tissues, antibody producing cells and antibodies. In certain embodiments, provided herein are methods for rapidly producing antibody producing organisms, tissues, cells and antibodies derived from humans, organisms, plants or cells that are genetically altered to over-express certain proteins.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.12/467,957, filed on May 18, 2009, which claims the benefit of U.S.Provisional Application No. 61/054,047, filed May 16, 2008, all of whichare incorporated herein by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Contract number R01 CA117802 by the National Cancer Institute(NCI) of the National Institute of Health (NIH).

BACKGROUND OF THE INVENTION

Traditional approaches to generate antigen specific monoclonalantibodies are limited. Typically only a small representation of thehighly diverse antigen-specific B-cell population is obtained. In somecases antigen specific B-cells fail to proliferate due toself-tolerance. In other cases, antigen specific B-cells fail toproliferate after cell fusion with a myeloma fusion partner.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are methods for generating,raising, obtaining and/or producing (for simplicity, the word“producing” as used herein is meant to equally refer to “generating,”“raising,” and “obtaining,”), a desired antibody are. In addition,systems, cell lines and organisms are described for use in producing adesired antibody. Further described are antibodies and compositionscomprising such antibodies.

Provided herein are various methods for producing (a) antibody producingcells and (b) antibodies that overcome many of the problems associatedwith conventional antibody production. In certain embodiments, providedherein are methods for producing (a) antibody producing cells and (b)antibodies derived from cells (e.g., mammalian cells) that are thatover-express MYC. In some embodiments, over-expression of MYC is inducedby contacting the cell with a small molecule, a biologic, a peptide, anantibody, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, provided herein is a method for (a)producing antibody producing cells and (b) antibodies derived from cellswherein MYC is over-expressed. In some embodiments, over-expression ofMYC is induced by contacting the cell with a small molecule, a biologic,a peptide, an antibody, or a combination thereof. In some embodiments,the small molecule is an antagonist of Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1. In some embodiments, the small molecule is anantagonist of Mxi-1. In some embodiments, the small molecule is anantagonist of MAD. In some embodiments, over-expression of MYC isinduced by contacting the cell with a siRNA molecule for Max-1, Mxi-1,MAD, or a combination thereof. In some embodiments, over-expression ofMYC is induced by contacting the cell with a shRNA molecule for Max-1,Mxi-1, MAD, or a combination thereof. In certain embodiments, providedherein is a method for producing an antibody from an immuno-deficientorganism (e.g., a mammal) wherein the organism's immune system isreconstituted with a plurality of hematopoietic stem cells that comprisean exogenous MYC gene (i.e., a MYC nucleic acid sequence (e.g., atransgenic sequence) that encodes a Myc peptide; i.e., transgenic MYC).Also provided herein is a method of producing a human or humanizedantibody by introducing (or otherwise providing to) a human gene thatencodes the human antibody into a cell (e.g., a mammalian cell) thatcomprises transgenic MYC. Certain embodiments, herein provide anisolated B-cell for producing a human or humanized antibody, wherein theB-cell over-expresses a MYC gene. In addition, provided herein is anisolated antibody prepared according to the any of the methods disclosedherein. Provided herein is also a cell that has been engineered toproduce an antibody prepared according any of the methods disclosedherein.

In certain embodiments, provided herein are methods for producingantibodies (e.g., monoclonal antibodies) without the need for cellfusion. In some embodiments, the methods disclosed herein producemonoclonal antibody without the need to fuse antibody producing B cellswith a fusion partner, thereby decreasing the time required to producethe antibody. Certain embodiments, described herein, provide for theproduction of antibodies specific for antigens that are normally subjectto immunological constraints (e.g., self tolerance). For example, insome embodiments, the methods are used to produce monoclonal antibodies(i.e., mAB) to self antigens.

Provided herein is a method for producing an antibody specific for anantigen, comprising contacting a cell comprising transgenic MYC with aselected antigen. In some embodiments, the cell (e.g., a mammalian cell)expresses CD79 on its cell surface. In some embodiments, the cell (e.g.,a mammalian cell) is a mammalian cell. In some embodiments, the cell(e.g., a mammalian cell) is a B-cell. In some embodiments, thetransgenic MYC gene comprises an inducible promoter or aB-cell-selective promoter operably linked to an open reading frame(i.e., ORF) of the gene. In some embodiments, the B-cell selectivepromoter is the Eμ promoter. In some embodiments, the inducible promotercomprises one or more TREs. In some embodiments, the cell (e.g., amammalian cell) is present in an organism (e.g., a mammal). In someembodiments, contacting the cell (e.g., a mammalian cell) with theselected antigen comprises administering (i.e., inoculating orintroducing) the selected antigen to the organism (e.g., mammal). Insome embodiments, the genome of the organism (e.g., a mammal) furthercomprises a nucleic acid sequence encoding a tetracycline reversetranscriptional activator (i.e., rtTA), a tetracycline transcriptionalactivator (i.e., tTA), or both. In some embodiments, a nucleic acidsequence encoding an rtTA comprises a B-cell-selective promoter operablylinked to an open reading frame of the sequence. In some embodiments, anucleic acid sequence encoding an rtTA comprises an MMTV promoteroperably linked to an open reading frame of the sequence. In someembodiments, a nucleic acid sequence encoding a tTA comprises aB-cell-selective promoter operably linked to an open reading frame ofthe sequence. In some embodiments, a nucleic acid sequence encoding atTA comprises an MMTV promoter operably linked to an open reading frameof the sequence.

In some embodiments, a method disclosed herein further comprisesproviding doxycycline, tetracycline, or an analog thereof to theorganism (e.g., a mammal) for a period to sufficient to suppresstTA-dependent expression or rtTA-dependent expression of the transgenicMYC gene. In some embodiments, a method disclosed herein furthercomprises (a) providing doxycycline, tetracycline, or an analog thereofto the organism (e.g., a mammal) for a period to sufficient to suppresstTA-dependent expression or rtTA-dependent expression of the transgenicMYC gene, and (b) withdrawing the doxycycline, tetracycline, or analogthereof after the period a time sufficient induce tTA-dependentexpression or rtTA-dependent expression of the transgenic MYC gene. Insome embodiments, the organism further comprises an exogenous nucleicacid sequence encoding the selected antigen. In some embodiments, themethod further comprises recovering from the organism one or moreB-cells that express an antibody specific for the selected antigen. Insome embodiments, the transgenic MYC gene encodes a Myc-ER polypeptide.In some embodiments, the transgenic MYC gene encodes a Myc-GRpolypeptide. As used herein, “GR” means glucocorticoid receptor. In someembodiments, a Myc-ER polypeptide is translocated to the nucleus of acell by contacting the cell (e.g., a mammalian cell) with an ER ligand.In some embodiments, the selected antigen is a self-antigen. In someembodiments, method comprises introducing (or otherwise providing to) anexpression vector encoding the selected antigen.

Provided herein is a method for producing an antibody specific for anantigen, comprising administering (i.e., introducing or inoculating) aselected antigen to an organism (e.g., a mammal), wherein the organism(e.g., a mammal) comprises a transgenic MYC gene and an induciblepromoter or a B-cell-selective promoter operably linked to an ORF of theMYC gene.

Provided herein is a method for producing an antibody specific for anantigen, comprising (a) providing a B-cell expressing an antibody thatspecifically binds to an antigen, wherein the B-cell comprises antransgenic MYC gene and/or an exogenous Myc peptide (e.g., a recombinantMyc peptide (e.g., a Myc fusion peptide as described herein)); and (b)inducing the expression of the MYC gene and/or the activity of the Mycpeptide (e.g., a recombinant Myc peptide (e.g., a Myc fusion peptide asdescribed herein)) in the B-cell. In some embodiments, the B-cell isprepared by contacting a B-cell comprising transgenic MYC gene and/or anexogenous Myc peptide (e.g., a recombinant Myc peptide (e.g., a Mycfusion peptide as described herein)) with the selected antigen. In someembodiments, a method disclosed herein further comprises expanding theB-cell to generate a monoclonal population of B-cells. In someembodiments, the method further comprises recovering the antibody fromthe population of monoclonal B-cells. In some embodiments, the B-cell ispresent in an organism (e.g., a mammal) and the induction of Mycactivity occurs in vivo. In some embodiments, a method disclosed hereinfurther comprises introducing (or otherwise providing to) a transgenicMYC gene or an exogenous Myc peptide (e.g., a recombinant Myc peptide(e.g., a Myc fusion peptide as described herein)) into the B-cell exvivo prior to the inducing step. In some embodiments, a method disclosedherein further comprises introducing (or otherwise providing to) anexogenous Myc peptide (e.g., a recombinant Myc peptide (e.g., a Mycfusion peptide as described herein)) into the B-cell ex vivo. In someembodiments, the exogenous Myc peptide (e.g., a recombinant Myc peptide(e.g., a Myc fusion peptide as described herein)) comprises a proteintransduction domain (e.g., a TAT domain). In some embodiments, theB-cell is an anergic B-cell. In some embodiments, the selected antigenis a self-antigen. In some embodiments, the transgenic MYC genecomprises a B-cell-selective promoter operably linked to an open readingframe of the gene. In some embodiments, the nucleic acid sequence (e.g.,a transgenic sequence) comprises an inducible promoter operably linkedto the open reading frame of the gene. In some embodiments, theinducible promoter comprises one or more TREs and the cell (e.g., amammalian cell) further expresses a tTA peptide or an rtTA peptide. Insome embodiments, the recombinant cell expresses the tTA peptide. Insome embodiments, the exogenous Myc peptide (e.g., a recombinant Mycpeptide (e.g., a Myc fusion peptide as described herein)) is a Myc-ERfusion peptide. In some embodiments, the inducing comprises contactingthe B-cell with an ER ligand. In some embodiments, the exogenous Mycpeptide (e.g., a recombinant Myc peptide (e.g., a Myc fusion peptide asdescribed herein)) is a Myc-GR polypeptide. In some embodiments, theinducing comprises contacting the B-cell with a GR ligand. In someembodiments, the B-cell is a mouse B-cell. In some embodiments, theB-cell is from an organism (e.g., a mammal) that was administered (i.e.,inoculated with or immunized with) the selected antigen. In someembodiments, the immunized organism comprises a transgenic MYC gene. Insome embodiments, the transgenic MYC gene is inducible. In someembodiments, the transgenic MYC gene comprises a promoter, and whereinthe promoter comprises one or more TREs. In some embodiments, theorganism further carries and expresses a tTA nucleic acid sequence(e.g., a transgenic sequence) or an rtTA nucleic acid sequence (e.g., atransgenic sequence). In some embodiments, the organism further carriesand expresses a gene (e.g., an exogenous gene) encoding the selectedantigen.

Provided herein is a method for producing an antibody that specificallybinds to an antigen, comprising administering to an immuno-deficientmammal a plurality of hematopoietic stem cells that comprise antransgenic MYC gene; and (c) administering (i.e., introducing, orinoculating) a selected antigen to the immuno-deficient mammal. In someembodiments, the hematopoietic stem cells further comprise an exogenousBCL-2 gene. In some embodiments, a method disclosed herein furthercomprises inducing a plurality of the hematopoietic stem cells todifferentiate into B-cells. In some embodiments, a method disclosedherein further comprises recovering a plurality of B-cells that expressthe antibody from the immuno-deficient mammal. In some embodiments, amethod disclosed herein further comprises recovering the antibody fromthe plurality of B-cells that express the antibody. In some embodiments,the transgenic MYC gene comprises an inducible promoter or aB-cell-selective promoter. In some embodiments, the transgenic MYC genecomprises an inducible promoter comprising one or more TREs. In someembodiments, the hematopoietic stem cells express tTA or rtTA. In someembodiments, a method disclosed herein further comprises (a) providingdoxycycline, tetracycline, or an analog thereof to the immuno-deficientmammal for a period of time sufficient to suppress the tTA-dependenttrans activation, and (b) withdrawing the doxycycline, tetracycline, oranalog thereof after the period time in order to allow tTA-dependenttransactivation. In some embodiments, the B-cell selective promoter isthe Eμ promoter. In some embodiments, the transgenic MYC gene encodes aMyc-ER polypeptide. In some embodiments, a method disclosed hereinfurther comprises (a) recovering at least one B-cell that express theantibody specific antigen from the immune-deficient mammal; and (b)contacting the B-cell with an ER ligand. In some embodiments, theexogenous Myc peptide (e.g., a recombinant Myc peptide (e.g., a Mycfusion peptide as described herein)) is a Myc-GR polypeptide. In someembodiments, a method disclosed herein further comprises (a) recoveringat least one B-cell that express the antibody specific antigen from theimmuno-deficient mammal; and (b) contacting the B-cell with a GR ligand.

In some embodiments, the selected antigen is a self-antigen. In someembodiments, immuno-deficient organism is obtained by irradiating theorganism (e.g., mammal). In some embodiments, the immuno-deficientorganism (e.g., mammal) is a Rag-1ko, Rag-2, SCID, DNA-PK, Ku70, Ku80,XRCC4, or μMT mouse. In some embodiments, the immuno-deficient organism(e.g., mammal) expresses the selected antigen. In some embodiments, theimmuno-deficient organism (e.g., mammal) comprises an exogenous DNAsequence that encodes the selected antigen. In some embodiments, theselected antigen is introduced into the organism's genome bytransfection with a nucleic acid expression vector or infection with aviral expression vector. In some embodiments, the expression vector is alentivirus.

Provided herein is a method for producing a human or humanized antibodycomprising: providing a cell (e.g., a mammalian cell) that expresseshuman antibodies and comprises an exogenous DNA sequence that encodes aMyc peptide (e.g., a recombinant Myc peptide (e.g., a Myc fusion peptideas described herein)); and (b) contacting the cell (e.g., a mammaliancell) with a selected antigen. In some embodiments, the cell (e.g., amammalian cell) is a B-cell. In some embodiments, the cell (e.g., amammalian cell) inducibly over-expresses Myc. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments,over-expression of MYC is induced by contacting the cell with a shRNAmolecule for Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the cell (e.g., a mammalian cell) is present in an organism(e.g., mammal). In some embodiments, the method further comprisesrecovering the cell (e.g., a mammalian cell) from the organism (e.g.,mammal). In some embodiments, the organism (e.g., mammal) is a mouse. Insome embodiments, the organism (e.g., mammal) is an MMTV-tTA/TRE-MYCmouse. In some embodiments, the organism (e.g., mammal) is obtained by:(a) presenting an immuno-deficient organism (e.g., mammal); and (b)administering to the organism (e.g., mammal) a plurality ofhematopoietic stem cells that over-express MYC. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments,over-expression of MYC is induced by contacting the cell with a shRNAmolecule for Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the immuno-deficient organism (e.g., mammal) is obtained byirradiating the organism (e.g., mammal). In some embodiments, theimmuno-deficient organism (e.g., mammal) is a Rag-1ko, Rag-2, SCID,DNA-PK, Ku70, Ku80, XRCC4, or μMT mouse. In some embodiments, theorganism (e.g., mammal) expresses the selected antigen. In someembodiments, a method disclosed herein further comprises recovering anantibody. In some embodiments, a method disclosed herein furthercomprises subjecting the cell (e.g., a mammalian cell) to conditionsthat induce over-expression of MYC. In some embodiments, over-expressionof MYC is induced by contacting the cell with a small molecule, abiologic, a peptide, an antibody, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1, Mxi-1, MAD,or a combination thereof. In some embodiments, the small molecule is anantagonist of Max-1. In some embodiments, the small molecule is anantagonist of Mxi-1. In some embodiments, the small molecule is anantagonist of MAD. In some embodiments, over-expression of MYC isinduced by contacting the cell with a siRNA molecule for Max-1, Mxi-1,MAD, or a combination thereof. In some embodiments, over-expression ofMYC is induced by contacting the cell with a shRNA molecule for Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the MYC DNAsequence comprises an inducible promoter or a B-cell-selective promoter.In some embodiments, prior to isolating the cell (e.g., a mammaliancell) from the organism (e.g., mammal) the cell (e.g., a mammalian cell)is subjected to conditions that induce over-expression of MYC. In someembodiments, over-expression of MYC is induced by contacting the cellwith a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.In some embodiments, over-expression of MYC is induced by contacting thecell with a shRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof.

In some embodiments, subsequent to isolating the antibody producing cellfrom the mammal the antibody producing cell is subjected to conditionsthat induce over-expression of MYC. In some embodiments, over-expressionof MYC is induced by contacting the cell with a small molecule, abiologic, a peptide, an antibody, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1, Mxi-1, MAD,or a combination thereof. In some embodiments, the small molecule is anantagonist of Max-1. In some embodiments, the small molecule is anantagonist of Mxi-1. In some embodiments, the small molecule is anantagonist of MAD. In some embodiments, over-expression of MYC isinduced by contacting the cell with a siRNA molecule for Max-1, Mxi-1,MAD, or a combination thereof. In some embodiments, over-expression ofMYC is induced by contacting the cell with a shRNA molecule for Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the mammal isan MMTV-rtTA/TRE-MYC; MMTV-rtTA/TRE-MYC/Rag-1−/−; MMTV-tTA/TRE-MYC; orMMTV-tTA/TRE-MYC/Rag-1−/− mouse and the conditions that induceover-expression of MYC are exposure to doxycycline, tetracycline, or ananalog thereof. In some embodiments, the Myc peptide is a Myc-ER fusionpeptide. In some embodiments, a method disclosed herein furthercomprises contacting the mammalian cell with an estrogen receptorligand. In some embodiments, the mammalian cell is contacted with theselected antigen in the absence of an estrogen receptor ligand. In someembodiments, the Myc peptide is a Myc-GR fusion peptide. In someembodiments, a method disclosed herein further comprises contacting themammalian cell with a glucocorticoid receptor ligand. In someembodiments, the mammalian cell is contacted with the selected antigenin the absence of an glucocorticoid receptor ligand.

Provided herein is a method for producing a human or humanized antibodycomprising: (a) providing an human cell; (b) isolating a human gene thatencodes the antibody from the human cell; and (c) introducing (orotherwise providing to) the gene into an cell that comprises antransgenic MYC gene or a exogenous Myc peptide (e.g., a recombinant Mycpeptide (e.g., a Myc fusion peptide as described herein)). In someembodiments, the human gene encodes human IgH (immunoglobulin heavychain) and IgL (immunoglobulin light chain), wherein the IgH and IgLtogether form an antibody that specifically binds the selected antigen.In some embodiments, the cell (e.g., a mammalian cell) is a B-cell. Insome embodiments, a method disclosed herein further comprisestransplanting the cell (e.g., a mammalian cell) into an organism (e.g.,a mouse). In some embodiments, the human gene isolated encodes a firstantibody and a second antibody. In some embodiments, a method disclosedherein further comprises recovering the antibody from the cell (e.g., amammalian cell). In some embodiments, the transgenic MYC DNA sequencecomprises an inducible promoter or a B-cell-selective promoter. In someembodiments, the exogenous Myc peptide (e.g., a recombinant Myc peptide(e.g., a Myc fusion peptide as described herein)) is a Myc-ER fusionpeptide. In some embodiments, the Myc peptide is a Myc-GR fusionpeptide.

Provided herein is a method for producing a human or humanized antibodycomprising: (a) introducing (or otherwise providing to) a gene encodinga human immunoglobulin into a cell that over-expresses Myc; and (b)isolating the encoded human immunoglobulin. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments,over-expression of MYC is induced by contacting the cell with a shRNAmolecule for Max-1, Mxi-1, MAD, or a combination thereof.

Provided herein are isolated B-cells for producing a human or humanizedantibody, wherein the B-cells over-expresses Myc. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments,over-expression of MYC is induced by contacting the cell with a shRNAmolecule for Max-1, Mxi-1, MAD, or a combination thereof. Also providedherein are isolated antibodies prepared according to the process of anyof the methods disclosed herein. And further provided herein aremammalian cells that have been engineered to produce a recombinant formof any antibody prepared according the process of the methods disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. An illustrative example of surface phenotype of tumors and celllines that arise in Eμ-MYC/BCR^(HEL)/sHEL transgenic mice. The blackfilled histograms correspond to the profiles of cells obtained from wildtype mice, the grey trace corresponds to the BCR^(HEL) transgenic mice,the thin line or dotted trace correspond to BCR^(HEL)/sHEL mice, and theblack trace correspond to the cells obtained from triply transgenicmice. The data represent the expression of the indicated markers onB220+ splenocytes.

FIG. 2. An illustrative example of immunoglobulin production and HELspecific titers in tumors and cell lines that arise inEμ-MYC/BCRHEL/sHEL mice. A defined number of cells (10⁵) derived fromeach cell line (TBL-1, TBL-8, TBL-14 and TTLN9, all derived from thetumors that arose in Eμ-MYC/BCR^(HEL)/sHEL mice) were seeded in a 24well plate, in 1 ml of growth medium, without any added cytokines.Samples of the supernatant were collected 4 days later and assayed forthe concentration of total IgM (panel A), as well as for the titer ofHEL specific IgM (panel B). Sera from various mice were used to compareantibody production to the cell lines. These mice included wild typeC57/BL6 mice (WT), BCR^(HEL) transgenic mice (BCR-tg), sHEL transgenicmice (Ag-tg), BCR^(HEL)/sHEL doubly transgenic mice (BCR/Ag-tg), Eμ-MYCmice and tumor-bearing Eμ-MYC/BCR^(HEL)/sHEL triply transgenic mice(BL). The results presented here are from one experiment, representativeof three independent assays.

FIG. 3. Appearance of activated B-cells following acute overexpressionof MYC. Flow cytometric detection of activated B-cells. Analyses wereperformed on lymph node cells obtained from a wild type mouse (blackfilled histogram), a MMTV-tTA/TRE-MYC/BCRHEL/sHEL mouse that had beenkept on doxycycline throughout (grey trace), and anMMTV-tTA/TRE-MYC/BCRHEL/sHEL mouse that had been taken off doxycycline aweek prior to euthanasia (black trace). Cells were stained withantibodies to two molecules that are upregulated following the selectedantigen-dependent activation of B-cells, CD69 (A), and B7-2 (CD86) (B).The traces represent the levels of CD69 and B7-2 present on the B220+fraction of the cells, ascertained by gating on the Cychrome-C stainingcells by flow cytometry.

FIG. 4. Accumulation of activated B-cells requires the continuousoverexpression of MYC. Activation of B-cells in response to MYC. Thenumber of activated B-cells in lymph nodes was determined as describedpreviously (34). Each data point in these graphs represents the numberof activated B-cells detected in the lymph nodes of an individual mouse.Cohorts of four mice were used for each time point. This figure showsthe requirement for MYC in the initiation and maintenance of theaccumulation of activated B-cells in inducedMMTV-tTA/TRE-MYC/BCRHEL/sHEL mice.

FIG. 5. Accumulation of autoantibodies in serum following theoverexpression of MYC. Serological evidence of broken tolerance. Serawere obtained from groups of four mice of each of the specifiedgenotypes, and assayed in triplicate by ELISA against HEL (A), or fortotal serum immunoglobulin (B). The numbered categories represent seraobtained from wild type mice (1), BCRHEL mice (2), BCRHEL/sHEL mice (3),Eμ-MYC/BCRHEL/sHEL mice prior to the development of overt tumors (4),MMTV-tTA/TRE-MYC/BCRHEL/sHEL mice that had been maintained ondoxycycline throughout (5), and MMTV-tTA/TRE-MYC/BCRHEL/sHEL mice thathad been taken off doxycycline 28 days prior to collection of sera (6).

FIG. 6. Accumulation of autoantibodies and immune complexes in thekidneys following the overexpression of MYC. Kidneys were obtained froma wild type mouse (A) or an Eμ-MYC/BCRHEL/sHEL mouse (B) forhistological examination. The tissues were sectioned and stained withhematoxylin and eosin, and microscopic images were obtained.Magnification was 100×. For immununofluorescence, kidneys were obtainedfrom a wild type mouse (C) or an Eμ-MYC/BCRHEL/sHEL mouse (D). Frozentissues were sectioned and stained with Rhodamine conjugated antibodiesto IgM, as described in Materials and Methods. Magnification was 5×.

FIG. 7. Protection of mice with novel HEL-specific antibodies fromlethal challenge of PRV variants that express HEL. Cohorts of mice wereinfected by intravenous administration of two different variants of PRVa Us9-GFP variant (solid line) or a Us9-HEL variant of the virus (dashedline). The viral supernatants were incubated with anti-HEL antibodies wedeveloped from MMTV-tTA/TRE-MYC/BCRHEL/sHEL mice for 1 hour, on ice,prior to injection into the animals. All animals were injected at thesame time, and examined three times a day for presentation of clinicalsigns consistent with PRV infection. This is one experimentrepresentative of two independent assays.

FIG. 8. Surface phenotype of B-cell tumors developed in retroviralchimeric mice. TRE-MYC bone marrow derived HSCs were transduced withpMIG-tTA and pMIG-H5. The transduced cells were used to reconstitutecohorts of lethally irradiated mice. The mice began to exhibit eternallyevident clinical signs of hematological malignancies and wereeuthanized. Lymph nodes and spleens were collected and used to generatesingle cell suspensions. A fraction of those cells were stained withantibodies to B220 and IgM. The panels represent the results of the flowcytometric analysis performed on those cells. The tumors were composedof mature, activated (blasting) B-cells, similar to what we observed inour mouse models of Burkitt's lymphoma, that yielded MYC-driven, antigendependent tumors composed of mature, activated B-cells.

FIG. 9. Western blot analysis of reactivity of serum obtained fromretroviral chimeric mice. TRE-MYC bone marrow derived HSCs weretransduced with pMIG-tTA and pMIG-H5. The transduced cells were used toreconstitute cohorts of lethally irradiated mice. The mice began toexhibit externally evident clinical signs of hematological malignanciesand were euthanized. Blood was collected shortly after euthanasia, andallowed to clot in order to obtain the serum. In order to testreactivity to H5 HA, 293FT cells were transiently transfected witheither pMIG, pcDNA2-H5 or pMIG-H5 (the same plasmid used to generate thechimeric mice). The cells were collected 48 hours after transfection andlysed with a Triton X-100 based lysis buffer, as previously described.The lysates were run on a 12% SDS-PAGE gel and transferred onto PVDFmembrane. The blots were probed with the serum obtained from theretroviral chimeric mice at a dilution of 1:5000. The lower molecularweight band (19Kd) is non-specific and serves as a good loading controlin this instance). Specific bands recognized by the serum represent thepredicted molecular weight for the uncleaved (HA0) H5 (˜60 kDa) and theHA1 subunit (˜40 kDa) that results from cleavage of HA0 by furinprotease during processing in the ER.

FIG. 10. Hemagglutination inhibition analysis of mouse serum obtainedfrom retroviral chimeric mice. Sera isolated from three mice (1-3), 6 to8 weeks after H5/tTA BM transduction, or phosphate buffered saline [PBS](C1) was diluted serially across microtiter plates in duplicate wells.Following serial dilution, 4 agglutinating units of influenza A virusesA/Mal/WI/944/82 (H5N2), A/NY/1469/02 (H1N1), or PBS (No virus) wereadded to each well and incubated for 30 min. Next turkey red blood cellswere added and incubated for 30 minutes to detect hemagglutinationactivity. The first column contains a final serum concentration of 1:20.

FIG. 11. Antibodies were obtained from mice immunized with HEL and CFA(Complete Freund's Adjuvant), or HEL and TAT-Myc. Antibodies obtainedfrom mice immunized with HEL and TAT-Myc are detected earlier andproduced a more robust response.

FIG. 12. Antibodies were obtained from mice immunized with HEL and CFA(Complete Freund's Adjuvant), or HEL and TAT-Myc on day 0. The mice wereadministered a booster shot of antigen on day 30. Mice immunized withHEL and TAT-Myc demonstrated improved host recall response to the HELantigen.

FIG. 13. Antibodies were obtained from mice immunized with Fluvirin(2007-2008) and CFA (Complete Freund's Adjuvant), or HEL and TAT-Myc.Antibodies obtained from mice immunized with Fluvirin and TAT-Myc aredetected earlier and produced a more robust response.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are systems and methods for generating antibodies with(a) improved specificity, (b) significantly increased potency, orcombination thereof. In some embodiments, the systems and methods aremore efficient and rapid than current approaches.

In some embodiments, the absence of self-tolerance enables these systemsto generate antibodies without significant (or any) immunologicalconstraints. The systems and approaches allow the generation of antibodyspecificities that cannot be accomplished by current immunizationprocedures.

In some embodiments, the cognate antigen drives B-cell neoplasia invivo, and allows for the selection of the antibody producing cells thatwill develop into lymphoma and the resulting monoclonal antibodyproducing cell lines. Such systems and approaches accelerate the timefor antibody development, as such systems and approaches allow screeningof the resulting monoclonal antibodies to be performed in a directedmanner.

In some embodiments, the methods and systems described herein providethe ability to generate and clonally expand antibody producing celllines from tumors, thus eliminating the need for cell fusion of theantibody producing splenic B-cells with a myeloma fusion partner. Thecell fusion process, described in the art, is fairly inefficient andonly allows for the immortalization of a fraction of the B-cells thatproliferate after immunization, and hence, limits the number and varietyof antigen specific monoclonal antibodies obtained.

CERTAIN DEFINITIONS

Unless indicated otherwise, the following terms have the followingmeanings when used herein and in the appended claims.

The term “leukocyte” comprises, by way of non-limiting example,lymphocytes, monocytes, macrophages, eosinophils, neutrophils andbasophils. In some embodiments, leukocytes refer to hematopoietic stemcells and all myeloid and lymphoid lineages that arise fromhematopoietic stem cells. In some embodiments, leukocytes refer allimmature, mature, undifferentiated and differentiated white blood cellpopulations including tissue specific and specialized varieties.

The term “lymphocyte” encompasses, by way of non-limiting example,B-cells, T-cells, NKT cells, and NK cells. In some embodiments,lymphocytes refers to all immature, mature, undifferentiated anddifferentiated white lymphocyte populations including tissue specificand specialized varieties. In some embodiments, lymphocytes include allB-cell lineages including pre-B-cells, Progenitor B cells, Early Pro-Bcells, Late Pro-B cells, Large Pre-B cells, Small Pre-B cells, ImmatureB cells, Mature B cells, plasma B-cells, memory B-cells, B-1 cells, B-2cells and anergic AN1/T3 cell populations.

The term B-cell, refers to, by way of non-limiting example, apre-B-cell, Progenitor B cell, Early Pro-B cell, Late Pro-B cell, LargePre-B cell, Small Pre-B cell, Immature B cell, Mature B cell, plasmaB-cell, memory B-cell, B-1 cell, B-2 cells and anergic AN1/T3 cellpopulations. In some embodiments, the term B-cell includes a B-cell thatexpresses an immunoglobulin heavy chain and/or light chain on its cellssurface. In some embodiments, the term B-cell includes a B-cell thatexpresses and secretes an immunoglobulin heavy chain and/or light chain.In some embodiments, the term B-cell includes a cell that binds anantigen on its cell-surface. In some embodiments, disclosed herein,B-cells or AN1/T3 cells are utilized in the processes described. Incertain embodiments, such cells are optionally substituted with anyanimal cell suitable for expressing, capable of expressing (e.g.,inducible expression), or capable of being differentiated into a cellsuitable for expressing an antibody including, e.g., a hematopoieticstem cell, a B-cell, a pre-B-cell, a Progenitor B cell, a Early Pro-Bcell, a Late Pro-B cell, a Large Pre-B cell, a Small Pre-B cell, anImmature B cell, a Mature B cell, a plasma B-cell, a memory B-cell, aB-1 cell, a B-2 cell, an anergic B-cell, or an anergic AN1/T3 cell.

The term “immunize” refers to the introduction of an antigen into anorganism by any suitable method. Non-limiting examples of various routesare by way of intradermal injection, intravenous injection, intraocularadministration, subcutaneous injection, intraperitoneal injection, oraladministration, or topical administration.

The term “antigen” refers to a substance that is capable of inducing theproduction of an antibody. In some embodiments, an antigen is asubstance that binds to an antibody variable region. In someembodiments, the selected antigen is a susbstance that is not native tothe antibody-producing organism. In some embodiments, the selectedantigen is a susbstance that is native to the antibody-producingorganism (e.g., a self antigen).

The term “AN1/T3 cell populations” refers to an anergic population ofB-cells; embodiments that describe AN1/T3 cell populations also includeembodiments, in which the term AN1/T3 cell populations is replaced withthe term “anergic population of B-cells.”

The term “anergic” refers to a CD79 expressing cell that is notactivated upon antigen binding. In some embodiments, activation isdefined by the up-regulation of cell-surface CD79. In some embodiments,activation is defined by phosphorylation of CD79a and/or phosphorylationof Syk. In some embodiments, activation is defined by calciummobilization.

The term “CD79” refers to the cell surface protein comprising eitherCD79a (Ig alpha) or CD79b (Ig beta).

The term “fusion protein” and “fusion peptide” are used interchangeablyand refer to a contiguous polypeptide chain comprising at least twodifferent proteins, parts of proteins or domains of proteins that arenot normally found together in nature.

The terms “Myc”, “cMyc”, “Myc protein” and “Myc polypeptide” are usedinterchangeably and refer in certain instances to the NCBI AccessionNumber NP002458.2, functional homologs, analogs or fragments thereof.Synonyms of Myc include, but are not limited to c-Myc, v-Myc, Mycproto-oncogene protein & Transcription factor p 64. In some embodiments,a Myc polypeptide comprises an amino acid sequence that is at least 40%to 100% identical, e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, orany other percent from about 40% to about 100% identical to the sequenceof NCBI Accession Numbers NP002458.2. In some embodiments, a Mycpolypeptide comprises a polypeptide sequence of 40 amino acids or morein length that is at least 50% to 100% identical, e.g., at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%,95%, 96%, 97%, 98%, or any other percent from about 50% to about 100%identical to the sequence of NCBI Accession Numbers NP002458.2. In someembodiments, a Myc polypeptide comprises a polypeptide sequence of 40amino acids or more in length that is at least 50% to 100% identical,e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about50% to about 100% identical to the sequence of NCBI Accession NumbersNP002458.2 wherein the Myc polypeptide promotes cell viability, cellimmortality, cell growth and/or cell proliferation. Furthermore, thefunction of an onco-peptide as used herein refers to one or more of thepromotion of cell viability, cell immortality, cell growth and/or cellproliferation. In several embodiments, disclosed herein, Myc is utilizedas an illustrative example of an onco-peptide with onco-peptidefunction. It is to be understood that in those embodiments, disclosedherein, the Myc is optionally substituted with any suitableonco-peptide, analog, homolog, or fragment thereof that promotes cellviability, cell immortality, cell growth and/or cell proliferation.

“MYC” refers to a nucleic acid encoding a Myc peptide (e.g., arecombinant Myc peptide (e.g., a Myc fusion peptide as describedherein)).

A MYC gene comprises a nucleotide sequence of at least 120 nucleotidesthat is at least 60% to 100% identical or homologous, e.g., at least 60,65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%,97%, 98%, or any other percent from about 70% to about 100% identical tosequences of NCBI Accession Number NM 002467.

“Myc-ER” refers to a Myc peptide fused to a modified hormone bindingdomain of the estrogen receptor (ER). When exposed to 4-hydroxytamoxifenor other estrogen analogs, the Myc-ER polypeptide is triggered totranslocate into the nucleus of the cell.

“Myc-ER” refers to a transgene encoding a Myc-ER polypeptide.

“Myc-GR” refers to a Myc peptide fused to a modified hormone bindingdomain of the glucocorticoid receptor (GR).

“MYC-GR” refers to a transgene encoding a Myc-GR polypeptide.

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences can be aligned for optimal comparisonpurposes (e.g., gaps are introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions can then becompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent homology between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In some embodiments, the two sequences are the samelength.

To determine percent homology between two sequences, the algorithm ofKarlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268,modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-5877 is used. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul, et al. (1990) J. Mol. Biol.215:403-410. BLAST nucleotide searches are performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules described or disclose herein.BLAST protein searches are performed with the XBLAST program, score=50,wordlength=3. To obtain gapped alignments for comparison purposes,Gapped BLAST is utilized as described in Altschul et al. (1997) NucleicAcids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) are used. See the website of the National Center forBiotechnology Information for further details (on the World Wide Web atncbi.nlm.nih.gov). Proteins suitable for use in the methods describedherein also includes proteins having between 1 to 15 amino acid changes,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acidsubstitutions, deletions, or additions, compared to the amino acidsequence of any protein described herein. In other embodiments, thealtered amino acid sequence is at least 75% identical, e.g., 77%, 80%,82%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of any protein inhibitor described herein. Suchsequence-variant proteins are suitable for the methods described hereinas long as the altered amino acid sequence retains sufficient biologicalactivity to be functional in the compositions and methods describedherein. Where amino acid substitutions are made, the substitutionsshould be conservative amino acid substitutions. Among the common aminoacids, for example, a “conservative amino acid substitution” isillustrated by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine. The BLOSUM62 table is an amino acidsubstitution matrix derived from about 2,000 local multiple alignmentsof protein sequence segments, representing highly conserved regions ofmore than 500 groups of related proteins (Henikoff et al (1992), Proc.Natl Acad. Sci. USA, 89:10915-10919). Accordingly, the BLOSUM62substitution frequencies are used to define conservative amino acidsubstitutions that, in some embodiments, are introduced into the aminoacid sequences described or disclosed herein. Although it is possible todesign amino acid substitutions based solely upon chemical properties(as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

The term “expression” refers to one or more of the following events: (1)production of RNA from a DNA sequence (e.g., by transcription) within acell; (2) processing of an RNA transcript (e.g., by splicing, editing,5′ cap formation, and/or 3′ end formation) within a cell; (3)translation of RNA into a polypeptide or protein within a cell; (4)post-translational modification of a polypeptide or protein within acell; (5) presentation of a polypeptide or protein on the cell surface;(6) secretion or release of a polypeptide or protein from a cell.

The term “over-expression”, refers to a higher level of expression whencompared to the endogenous level of expression of an identicalpolypeptide or protein within the same cell. In certain instances,“over-expression” refers to recombinant expression of a polypeptide. Insome embodiments, a higher level of expression comprises 2% to 200%higher. In some embodiments, a higher level of expression comprises2-fold to 1000-fold higher. In some embodiments, a higher level ofexpression comprises 2-fold to 1000-fold higher. In some embodiments, ahigher level of expression comprises 2-fold to 10,000-fold higher. Insome embodiments, a higher level of expression comprises a detectablelevel of expression when compared to a previous undetectable level ofexpression. In some embodiments, “over-expression” refers to anydetectable level of expression of an exogenous polypeptide or protein.

The phrase “over-expression of MYC” refers to over-expression of a Mycpeptide (e.g., a recombinant Myc peptide (e.g., a Myc fusion peptide asdescribed herein)) or a Myc peptide (e.g., a recombinant Myc peptide(e.g., a Myc fusion peptide as described herein)) fused to anotherpeptide. In some embodiments, “over-expression of MYC” refers toover-expression of MYC-ER. In some embodiments, “over-expression of MYC”refers to over-expression of MYC-GR. In some embodiments,“over-expression of MYC” refers to over-expression of a fragment of aMyc-polypeptide that contains the DNA binding domain of Myc. In someembodiments, “over-expression of MYC” refers to over-expression of apolypeptide that comprises the DNA binding domain of Myc.

The terms “antibody” and “antibodies” refer to monoclonal antibodies,polyclonal antibodies, bi-specific antibodies, multispecific antibodies,grafted antibodies, human antibodies, humanized antibodies, syntheticantibodies, chimeric antibodies, camelized antibodies, single-chain Fvs(scFv), single chain antibodies, Fab fragments, Flab′) fragments,disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id)antibodies and antigen-binding fragments of any of the above. Inparticular, antibodies include immunoglobulin molecules andimmunologically active fragments of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site Immunoglobulin moleculesare of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass. The terms “antibody”and immunoglobulin are used interchangeably in the broadest sense. Thesubunit structures and three-dimensional configurations of the differentclasses of immunoglobulins are well known in the art. In someembodiments, an antibody is part of a larger molecule, formed bycovalent or non-covalent association of the antibody with one or moreother proteins or peptides.

The term “derivative” in the context of a polypeptide or protein, e.g.an antibody, refers to a polypeptide or protein that comprises an aminoacid sequence which has been altered by the introduction of amino acidresidue substitutions, deletions or additions. The term “derivative”also refers to a polypeptide or protein which has been modified, i.e.,by the covalent attachment of any type of molecule to the antibody. Forexample, in some embodiments, a polypeptide or protein is modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. In someembodiments, derivatives, polypeptides or proteins are produced bychemical modifications using suitable techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. In some embodiments, aderivative a polypeptide or protein possesses a similar or identicalfunction as the polypeptide or protein from which it was derived.

The terms “full length antibody”, “intact antibody” and “whole antibody”are used herein interchangeably, to refer to an antibody in itssubstantially intact form, and not antibody fragments as defined below.These terms particularly refer to an antibody with heavy chains containsFc regions. In some embodiments, an antibody variant provided herein isa full length antibody. In some embodiments, the full length antibody ishuman, humanized, chimeric, and/or affinity matured.

An “affinity matured” antibody is one having one or more alteration inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by suitable procedures. See,for example, Marks et al., (1992) Biotechnology 10:779-783 thatdescribes affinity maturation by variable heavy chain (VH) and variablelight chain (VL) domain shuffling. Random mutagenesis of CDR and/orframework residues is described in: Barbas, et al. (1994) Proc. Nat.Acad. Sci, USA 91:3809-3813; Shier et al., (1995) Gene 169:147-155;Yelton et al., 1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J.Immunol. 154(7):3310-9; and Hawkins et al, (19920, J. Mol. Biol.226:889-896, for example.

The terms “binding fragment”, “antibody fragment” or “antigen bindingfragment” are used herein, for purposes of the specification and claims,to mean a portion or fragment of an intact antibody molecule, preferablywherein the fragment retains antigen-binding function. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, Fd, Fd′ and Fv fragments,diabodies, linear antibodies (Zapata et al. (1995) Protein Eng. 10:1057), single-chain antibody molecules, single-chain bindingpolypeptides, scFv, bivalent scFv, tetravalent scFv, and bispecific ormultispecific antibodies formed from antibody fragments.

“Fab” fragments are typically produced by papain digestion of antibodiesresulting in the production of two identical antigen-binding fragments,each with a single antigen-binding site and a residual “Fc” fragment.Pepsin treatment yields a F(ab′)2 fragment that has twoantigen-combining sites capable of cross-linking antigen. An “Fv” is theminimum antibody fragment that contains a complete antigen recognitionand binding site. In a two-chain Fv species, this region consists of adimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain are covalently linked by aflexible peptide linker such that the light and heavy chains associatein a “dimeric” structure analogous to that in a two-chain Fv species. Itis in this configuration that the three CDRs of each variable domaininteract to define an antigen-binding site on the surface of the VH-VLdimer. Collectively, the six CDRs confer antigen-binding specificity tothe antibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although usually at a lower affinity thanthe entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H)1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy-chain C_(H)1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments that have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also suitable.Methods for producing the various fragments from monoclonal Abs include,e.g., Plückthun, 1992, Immunol. Rev. 130:152-188.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations that are present in minor amounts. In someembodiments, monoclonal antibodies are made, for example, by thehybridoma method first described by Köhler and Milstein (1975) Nature256:495, or are made by recombinant methods, e.g., as described in U.S.Pat. No. 4,816,567. In some embodiments, monoclonal antibodies areisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991), as well as in Marks et al.,J. Mol. Biol. 222:581-597 (1991).

The antibodies herein include monoclonal, polyclonal, recombinant,chimeric, humanized, bi-specific, grafted, human, and fragments thereofincluding antibodies altered by any means to be less immunogenic inhumans. Thus, for example, the monoclonal antibodies and fragments,etc., herein include “chimeric” antibodies and “humanized” antibodies.In general, chimeric antibodies include a portion of the heavy and/orlight chain that is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, so long as they exhibit the desired biologicalactivity (U.S. Pat. No. 4,816,567); Morrison et al. Proc. Natl Acad.Sci. 81:6851-6855 (1984). For example in some embodiments, a chimericantibody contains variable regions derived from a mouse and constantregions derived from human in which the constant region containssequences homologous to both human IgG2 and human IgG4. Numerous methodsfor preparing “chimeric” antibodies, etc., are known in the art.“Humanized” forms of non-human (e.g., murine) antibodies or fragmentsare chimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include, grafted antibodies or CDRgrafted antibodies wherein part or all of the amino acid sequence of oneor more complementarity determining regions (CDRs) derived from anon-human animal antibody is grafted to an appropriate position of ahuman antibody while maintaining the desired binding specificity and/oraffinity of the original non-human antibody. In some embodiments,corresponding non-human residues replace Fv framework residues of thehuman immunoglobulin. In some embodiments, humanized antibodies compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences. These modifications are made tofurther refine and optimize antibody performance. In some embodiments,the humanized antibody comprises substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. For further details, see, e.g.: Joneset al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332:323-329 (1988) and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).Numerous methods for “humanizing” antibodies, etc., are known in theart.

The term “vector” or “expression vector” is used herein to mean vectorsused as suitable vehicles for introducing (or otherwise providing to)into and/or expressing a desired gene in a host cell. Examples ofvectors include but are not limited to plasmids, phages, viruses andretroviruses. In some embodiments, suitable vectors comprise a selectionmarker. In some embodiments, suitable vectors comprise restriction sitesto facilitate cloning of the desired gene and in some embodiments,suitable vectors comprise the ability to enter and/or replicate ineukaryotic or prokaryotic cells.

Numerous expression vector systems are optionally utilized in a methoddisclosed herein. For example, one class of vector utilizes DNA elementsthat are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites. In someembodiments, cells that have integrated exogenous DNA into theirchromosomes are selected by introducing (or otherwise providing to) oneor more selection markers into the transfected host cells. In someembodiments, the selection markers provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. In some embodiments, the selectablemarker gene is either directly linked to the DNA sequences to beexpressed, or is introduced into the same cell by cotransformation. Insome embodiments, additional regulatory elements are incorporated in thevector for optimal transcription. Examples of regulatory elementsinclude signal sequences, splice signals, as well as transcriptionalpromoters, enhancers, and termination signals.

In some embodiments, the nucleic acids described herein include apromoter comprising a tetracycline response element and a transgene thatconstitutively expresses either rtTA (reverse tetracyclinetransactivator) or tTA (tetracycline-controlled transactivator). ThertTA protein binds to a TRE and activates transcription only in thepresence of tetracycline, doxycycline or an analogue thereof. The tTAprotein, in the presence of tetracycline, doxycycline or an analoguethereof, binds to a TRE thereby inhibiting transcription. In the absenceof tetracycline, doxycycline or an analogue thereof tTA allowstranscription.

The term “epitope” refers to a fragment of a polypeptide or proteinhaving antigenic or immunogenic activity in an organism, preferably in amammal, and most preferably in a human. An epitope having immunogenicactivity is a fragment of a polypeptide or protein that elicits anantibody response in an animal. An epitope having antigenic activity isa fragment of a polypeptide or protein to which an antibodyimmunospecifically binds as determined by any known method, for exampleby immunoassays. Antigenic epitopes need not necessarily be immunogenic.

The phrase “specifically binds” when referring to the interactionbetween an antibody or other binding molecule and a protein orpolypeptide or epitope, typically refers to an antibody or other bindingmolecule that recognizes and detectably binds with high affinity to thetarget of interest. Preferably, under designated or physiologicalconditions, the specified antibodies or binding molecules bind to aparticular polypeptide, protein or epitope yet does not bind in asignificant or undesirable amount to other molecules present in asample. In other words the specified antibody or binding molecule doesnot undesirably cross-react with non-target antigens and/or epitopes. Avariety of immunoassay formats are used to select antibodies or otherbinding molecule that are immunoreactive with a particular polypeptideand have a desired specificity. For example, solid-phase ELISAimmunoassays, BIAcore, flow cytometry and radioimmunoassays are used toselect monoclonal antibodies having a desired immunoreactivity andspecificity. See, Harlow, 1988, ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York (hereinafter, “Harlow”), for adescription of immunoassay formats and conditions that are used todetermine or assess immunoreactivity and specificity.

“Selective binding”, “selectivity”, and the like refer the preference ofan antibody to interact with one molecule as compared to another.Preferably, interactions between antibodies, particularly modulators,and proteins are both specific and selective. Note that in someembodiments, an antibody is designed to “specifically bind” and“selectively bind” two distinct, yet similar targets without binding toother undesirable targets.

The term “endogenous” in the context of a cellular protein refers toprotein naturally occurring and/or expressed by the cell in the absenceof recombinant manipulation; accordingly, the terms “endogenouslyexpressed protein” or “endogenous protein” excludes cellular proteinsexpressed by means of recombinant technology.

The terms “polypeptide”, peptide” and “protein” are used interchangeablyherein to refer to a polymer of amino acid residues. The terms apply tonaturally occurring amino acid polymers as well as amino acid polymersin which one or more amino acid residues is a non-naturally occurringamino acid, e.g., an amino acid analog. The terms encompass amino acidchains of any length, including full length proteins (i.e., antigens),wherein the amino acid residues are linked by covalent peptide bonds.

The term “onco-peptide” and “oncoprotein” are utilized interchangeablyherein and refer to a polymer of amino acid residues, fragments oranalogs thereof, that are encoded by oncogenes or proto-oncogenes. Incertain instances, onco-peptides promote cell survival and/or cellproliferation.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine Amino acidanalogs refers to agents that have the same basic chemical structure asa naturally occurring amino acid, i.e., an a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions are achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)).

The terms “isolated” and “purified” refer to a material that issubstantially or essentially removed from or concentrated in its naturalenvironment. For example, an isolated nucleic acid is one that isseparated from at least some of the nucleic acids that normally flank itor other nucleic acids or components (proteins, lipids, etc. . . . ) ina sample. In another example, a polypeptide is purified if it issubstantially removed from or concentrated in its natural environment.Methods for purification and isolation of nucleic acids and proteins aredocumented methodologies. Embodiments, of “substantially” include atleast 20%, at least 40%, at least 50%, at least 75%, at least 85%, atleast 90%, at least 95%, or at least 99%.

The phrase “recombinant nucleic acid” refers to a nucleic acid that isengineered through the combination or insertion of one or more nucleicacids, thereby combining sequences that would not normally occurtogether in nature. In some embodiments, recombinant nucleic acidscomprise promoters or enhances. In some embodiments, recombinant nucleicacids comprise restriction enzyme sites. In some embodiments,recombinant nucleic acids encode polypeptides. In some embodiments,recombinant nucleic acids comprise mutations.

The term “recombinant polypeptide” refers to a polypeptide that isproduced from a recombinant nucleic acid. In some embodiments, therecombinant polypeptide is a Myc fusion peptide. In some embodiments,the recombinant polypeptide is a TAT-Myc fusion peptide as describedherein.

The term “recombinant Myc polypeptide” comprises a Myc polypeptide thatis produced from a recombinant nucleic acid. In some embodiments, therecombinant Myc polypeptide is a Myc fusion peptide. In someembodiments, the recombinant Myc polypeptide is a TAT-Myc fusion peptideas described herein.

The term “recombinant Myc activity” refers to the binding of arecombinant Myc polypeptide to DNA located in the nucleus of the cellwherein the recombinant Myc regulates the transcriptional activity ofMyc responsive genes.

The term “transgene” refers to an exogenous gene introduced into thegenome of an organism.

The term “transgenic animal” refers to an animal that carries atransgene.

The term “genetically altered” refers to an animal, bacteria, virus orcell comprising a recombinant nucleic acid.

The term “self antigen” refers to an antigen that originates from withinan animal, tissue, or cell. In some embodiments, a self antigencomprises an endogenous antigen. In some embodiments, a self antigencomprises an endogenous antigen produced by an endogenous retrovirus. Insome embodiments, self antigens comprise neo-self antigens, microbiallyor parasite encoded neo-self antigens, or other neo-self antigensexpressed as a result of genetic alteration to an animal or cell. Insome embodiments, a chimeric mouse expresses a neo-self antigen.

The term “auto antigen” refers to an antigen that comprises an epitopeof a self antigen or an immunologically reactive epitope that mimicsthat of a self antigen. In some embodiments, the term auto antigencomprises antigens to which autoantibodies are produced. In someembodiments, an auto antigen comprises an endogenous antigen wherein theanimal from which the endogenous antigen originated is or was onceimmunologically tolerant to the selected antigen.

The term “neo-self antigen” refers to an antigen that is introduced intoan organism via use of a retrovirus. In some embodiments, a retrovirusis used to overexpress a protein in a stem cell and those cells aretransplanted into an organism. In some embodiments, the protein isviewed by the chimaeric's animal immunue system as a self antigen.

The term “Myc activity” refers to binding of a Myc peptide (e.g., arecombinant Myc peptide (e.g., a Myc fusion peptide as describedherein)) to DNA in the nucleus of a cell wherein Myc regulates thetranscriptional activity of Myc responsive genes. In some embodiments,Myc activity induces cell proliferation and/or antibody production.

The term “activation of Myc” and “Myc activation” refers to theinduction of Myc activity. In some embodiments, activation of Myc isinduced by over-expression of a Myc peptide (e.g., a recombinant Mycpeptide (e.g., a Myc fusion peptide as described herein)). In someembodiments, activation of Myc is induced by transport of a Myc peptide(e.g., a recombinant Myc peptide (e.g., a Myc fusion peptide asdescribed herein)) into the nucleus of a cell. In some embodiments,activation of Myc is induced by transport of a Myc peptide (e.g., arecombinant Myc peptide (e.g., a Myc fusion peptide as describedherein)) into a cell.

The term “TRE” refers to a tetracycline response element.

The term “immortal” refers the ability of a cell to proliferate inculture over multiple generations with a minimal loss of viability ofthe overall population. In some embodiments, immortal cells areconsidered transformed cells. In some embodiments, immortal cells areconsidered malignant. In some embodiments, immortal B-cell are referredto as lymphomas. In the absence of genetic alteration, mutation,modification, or viral infection, native primary cells and primaryB-cells are, in some embodiments, not immortal and will lose viabilityafter a few passages in culture.

As used herein, “xenomouse” means a mouse that is genetically altered toexpress one or more non-native genes. In some embodiments, a xenomouseis genetically altered to produce human antibodies.

“Transporter peptide” and “peptide transduction domain” (PTD) areinterchangeable. As used herein, the terms mean a peptide sequence thatpromotes peptide penetration into cells and tissues. In someembodiments, a transporter peptide is TAT. In some embodiments, atransporter peptide is TAT_([48-57]). In some embodiments, a transporterpeptide is TAT_([57-48]). In some embodiments, the transporter peptideis HIV-Vpr, HSV-Vp22, antennapedia, the chariot system, or a combinationthereof. For examples of transporter peptides, see U.S. application Ser.No. 11/583,970 (Pub. No. 2007-0116691), which is herein incorporated byreference for such disclosures.

As used herein, a “MYC sequence” is a MYC amino acid peptide sequence.In some embodiments, the MYC peptide is a complete MYC peptide sequence.In some embodiments, the MYC peptide is a partial MYC peptide sequence.In some embodiments, the MYC is c-MYC. In some embodiments, the MYCpeptide sequence comprises:

(SEQ ID NO. 1) MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSS LYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSA GGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRD QIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQ LR KGELNSKLE.

Processes for Preparing Antibodies

In some embodiments, provided herein is a method of producing anantibody that specifically binds to an antigen, comprising: contacting acell comprising a nucleic acid sequence (e.g., a transgenic sequence)encoding an onco-peptide (e.g., MYC) or an onco-peptide (e.g.,recombinant onco-peptide; e.g., TAT-Myc) with a selected antigen. Insome embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide is a fusion peptide comprising a PTD. Insome embodiments, the onco-peptide is TAT-MYC. In some embodiments, theonco-peptide is Myc-ER. In some embodiments, the onco-peptide is Myc-GR.

In some embodiments, the onco-peptide is a peptide that promotes cellsurvival and/or proliferation. In certain embodiments, the cell is amammalian cell. In some embodiments, the cell is a human cell. In someembodiments, the cell is a B-cell. In some embodiments, the cell is ahematopoietic cell. In some embodiments, the cell is a human B-cell. Insome embodiments, the cell is a human hematopoietic cell. In someembodiments, the cell (e.g., a mammalian cell) expresses CD79 on itscell surface. In some embodiments, the cell (e.g., a mammalian cell)comprises an intact salvage pathway for purine biosynthesis and istolerant and or anergic to the selected antigen. In some embodiments,the cell (e.g., a mammalian cell) is a B-cell, a B-cell precursor orprogenitor, or a hematopoietic stem cell. In certain embodiments, theantibody specifically binds to the selected antigen. In certainembodiments, the onco-peptide is a peptide that promotes cell survivaland/or proliferation.

In certain embodiments, the cell (e.g., a mammalian cell) is contactedwith the selected antigen by any suitable method. In specificembodiments, the cell (e.g., a mammalian cell) comprises a nucleic acidsequence (e.g., a transgenic sequence) that encodes the selected antigenand is, thereby, contacted with the selected antigen. In someembodiments, the selected antigen is a self-antigen.

In certain embodiments, wherein a B-cell precursor or progenitor or ahematopoietic stem cell are used, a method described herein furthercomprises allowing or inducing differentiation of the cell (e.g., amammalian cell) to a B-cell.

In some embodiments, a MYC sequence (e.g., a transgenic sequence)further comprises a B-cell selective promoter or an inducible promoter.In some embodiments, a nucleic acid sequence (e.g., a transgenicsequence) encoding the selected antigen further comprises a B-cellselective promoter or an inducible promoter. In some embodiments, aB-cell selective promoter is, by way of non-limiting example, the Eμpromoter. In some embodiments, the inducible promoter comprises, by wayof non-limiting example, one or more TREs. In certain embodiments, thecell (e.g., a mammalian cell) further comprises a nucleic acid sequence(e.g., a transgenic sequence) that encodes a nucleic acid sequence(e.g., a transgenic sequence) encoding a tTA peptide or an rtTA peptide.In some embodiments, the nucleic acid sequence (e.g., a transgenicsequence) encoding the tTA peptide or the rtTA peptide comprises aB-cell-selective promoter operably linked to the open reading frameencoding the tTA peptide or the rtTA peptide. In some embodiments, thenucleic acid sequence (e.g., transgenic nucleic acid sequence) encodingthe tTA peptide or the rtTA peptide comprises the MMTV promoter operablylinked to the open reading frame encoding the tTA peptide or the rtTApeptide. Furthermore, in some embodiments, wherein an inducible promotercomprises one or more TREs, a method described herein further comprisescontacting the cell (e.g., a mammalian cell) with doxycycline,tetracycline, or an analog thereof.

In some embodiments, the onco-peptide is a fusion peptide that comprisesa receptor that activates the onco-peptide activity (e.g., cell survivaland/or proliferation) when bound with a ligand. In some embodiments, thereceptor is an estrogen receptor (ER). In specific embodiments, theonco-peptide is a Myc-ER fusion peptide. In specific embodiments, theonco-peptide is a Myc-GR fusion peptide. In certain embodiments, amethod described herein further comprises contacting a cell comprising arecombinant onco-peptide that is a fusion peptide comprising a receptorwith a ligand that binds the receptor. In specific embodiments, a methoddescribed herein further comprises contacting the cell (e.g., amammalian cell) comprising a Myc-ER fusion peptide with an estrogenreceptor modulator (e.g., an ER agonist or antagonist). In specificembodiments, a method described herein further comprises contacting thecell (e.g., a mammalian cell) comprising a Myc-GR fusion peptide with aglucocorticoid receptor modulator (e.g., a GR agonist or antagonist).

In some embodiments, the onco-peptide is a fusion peptide that comprisesa (a) a transporter peptide sequence (e.g., TAT); and (b) a MYC sequence(e.g., c-MYC). In some embodiments, the fusion peptide is a peptide ofFormula (I):

transporter peptide sequence-MYC sequence.

In some embodiments, a fusion peptide disclosed herein comprises (a) atransporter peptide sequence; (b) a MYC sequence; and (c) one or moremolecules that link the transporter peptide sequence and the MYCsequence (i.e., “X”). In some embodiments, the fusion peptide is apeptide of Formula (II):

transporter peptide sequence-X-MYC sequence,

wherein -X- is molecule that links the transporter peptide sequence andthe MYC sequence. In some embodiments, -X- is an amino acid. In someembodiments, -X- is at least one amino acid.

In certain embodiments, a method described herein further comprisesinducing MYC expression, Myc activity, or a combination thereof, in thecell (e.g., a mammalian cell). In some embodiments, a method describedherein further comprises proliferating or inducing proliferation of thecell (e.g., a mammalian cell). In certain embodiments, the cells (e.g.,a mammalian cell) are proliferated at least until forming a lymphoma(e.g., a B-cell lymphoma). In some embodiments, the proliferating cellsare lymphoma cells. In certain embodiments, the cells (e.g., a mammaliancell) do not require and/or are not fused, e.g., with a myeloma, priorto proliferation (e.g., in order to provide a cell population sufficientto produce a significant or therapeutic amount of the antibody in areasonable period of time).

In some embodiments, the method further comprises recovering from thecell (e.g., a mammalian cell) the antibody that specifically binds theselected antigen. In some embodiments, the antibody produced is solubleand is not membrane bound.

In some embodiments, the cell (e.g., a mammalian cell) (e.g., amammalian cell) is present in an organism (e.g., a mammal). In someembodiments, the organism is a xenomouse. In certain embodiments, theorganism (e.g., a mammal) further comprises a nucleic acid sequence(e.g., a transgenic sequence) that encodes a transgenic onco-peptidesequence (e.g., MYC). In some embodiments, the organism (e.g., a mammal)further comprises a transgenic nucleic acid sequence encoding a tTApeptide or an rtTA peptide. In some embodiments, the nucleic acidsequence (e.g., transgenic nucleic acid sequence) encoding the tTApeptide or the rtTA peptide comprises a B-cell-selective promoteroperably linked to the open reading frame encoding the tTA peptide orthe rtTA peptide. In some embodiments, the nucleic acid sequence (e.g.,transgenic nucleic acid sequence) encoding the tTA peptide or the rtTApeptide comprises the MMTV promoter operably linked to the open readingframe encoding the tTA peptide or the rtTA peptide.

In certain embodiments, contacting a cell comprising a Myc peptide(e.g., a recombinant Myc peptide (e.g., a Myc fusion peptide asdescribed herein)) comprises introducing (or otherwise providing to) theselected antigen into the organism (e.g., a mammal) by any suitablemanner. In some embodiments, the onco-peptide is a fusion peptide. Insome embodiments, the onco-peptide is a fusion peptide comprising a PTD.In some embodiments, the onco-peptide is TAT-MYC. In some embodiments,the onco-peptide is Myc-ER. In some embodiments, the onco-peptide isMyc-GR. In some embodiments, the organism is a xenomouse. In certainembodiments, the organism (e.g., a mammal) further comprises a nucleicacid sequence (e.g., transgenic nucleic acid sequence) encoding theselected antigen. In some embodiments, the nucleic acid sequence (e.g.,transgenic nucleic acid sequence) encoding the selected antigencomprises a B-cell-selective promoter operably linked to the openreading frame encoding the selected antigen.

In some embodiments, a method described herein further comprisesproviding doxycycline, tetracycline, or an analog thereof to theorganism (e.g., a mammal) to suppress tTA-dependent expression of theMyc peptide. In some embodiments, a method described herein furthercomprises providing doxycycline, tetracycline, or an analog thereof tothe organism (e.g., a mammal) for a period of time sufficient tosuppress tTA-dependent expression of the transgenic MYC gene, andwithdrawing the doxycycline, tetracycline, or analog thereof after theperiod of time to induce tTA-dependent expression of the transgenic MYC.

In some embodiments, a method described herein further comprisesrecovering from the organism (e.g., a mammal; e.g., a xenomouse) a cell(e.g., B-cells) that express the antibody that specifically binds theselected antigen. In certain embodiments, a method described hereinfurther comprises recovering from the cells (e.g., mammalian cells)(e.g., B-cells) the antibody that specifically binds the selectedantigen. In some embodiments, a method described herein furthercomprises recovering from the organism (e.g., a mammal) the antibodythat specifically binds the selected antigen.

In some embodiments, provided herein is a method of producing anantibody that specifically binds to an antigen, comprising: contacting acell with a selected antigen and an onco-peptide (e.g. Myc).

In some embodiments, the onco-peptide is a peptide that promotes cellsurvival and/or proliferation. In certain embodiments, the cell is amammalian cell. In some embodiments, the cell is a human cell. In someembodiments, the cell is a B-cell. In some embodiments, the cell is ahematopoietic cell. In some embodiments, the cell is a human B-cell. Insome embodiments, the cell is a human hematopoietic cell. In someembodiments, the cell (e.g., a mammalian cell) expresses CD79 on itscell surface. In some embodiments, the cell (e.g., a mammalian cell)comprises an intact salvage pathway for purine biosynthesis and istolerant and or anergic to the selected antigen. In some embodiments,the cell (e.g., a mammalian cell) is a B-cell, a B-cell precursor orprogenitor, or a hematopoietic stem cell. In certain embodiments, theantibody specifically binds to the selected antigen. In certainembodiments, the onco-peptide is a peptide that promotes cell survivaland/or proliferation.

In certain embodiments, the cell (e.g., a mammalian cell) is contactedwith the selected antigen by any suitable method. In specificembodiments, the cell (e.g., a mammalian cell) comprises a nucleic acidsequence (e.g., a transgenic sequence) that encodes the selected antigenand is, thereby, contacted with the selected antigen. In someembodiments, the selected antigen is a self-antigen.

In certain embodiments, wherein a B-cell precursor or progenitor or ahematopoietic stem cell are used, a method described herein furthercomprises allowing or inducing differentiation of the cell (e.g., amammalian cell) to a B-cell.

In some embodiments, the onco-peptide is a fusion peptide that comprisesa receptor that activates the onco-peptide activity (e.g., cell survivaland/or proliferation) when bound with a ligand. In some embodiments, thereceptor is an estrogen receptor (ER). In some embodiments, the receptoris a glucocorticoid receptor (GR). In specific embodiments, theonco-peptide is a Myc-ER fusion peptide. In some embodiments, theonco-peptide is a Myc-GR fusion peptide. In certain embodiments, amethod described herein further comprises contacting a cell comprising arecombinant onco-peptide that is a fusion peptide comprising a receptorwith a ligand that binds the receptor. In specific embodiments, a methoddescribed herein further comprises contacting the cell (e.g., amammalian cell) comprising a Myc-ER fusion peptide with an estrogenreceptor modulator (e.g., an ER agonist or antagonist). In specificembodiments, a method described herein further comprises contacting thecell (e.g., a mammalian cell) comprising a Myc-GR fusion peptide with anglucocorticoid receptor modulator (e.g., a GR agonist or antagonist).

In some embodiments, the onco-peptide is a fusion peptide that comprisesa (a) a transporter peptide sequence (e.g., TAT); and (b) a MYC sequence(e.g., c-MYC). In some embodiments, the fusion peptide is a peptide ofFormula (I):

transporter peptide sequence-MYC sequence.

In some embodiments, a fusion peptide disclosed herein comprises (a) atransporter peptide sequence; (b) a MYC sequence; and (c) one or moremolecules that link the transporter peptide sequence and the MYCsequence (i.e., “X”). In some embodiments, the fusion peptide is apeptide of Formula (II):

transporter peptide sequence-X-MYC sequence,

wherein -X- is molecule that links the transporter peptide sequence andthe MYC sequence. In some embodiments, -X- is an amino acid. In someembodiments, -X- is at least one amino acid.

In some embodiments, the method further comprises recovering from thecell (e.g., a mammalian cell) the antibody that specifically binds theselected antigen. In some embodiments, the antibody produced is solubleand is not membrane bound.

In certain embodiments, contacting a cell comprising a Myc peptide(e.g., a recombinant Myc peptide, a TAT-Myc fusion peptide) comprisesintroducing (or otherwise providing to) the selected antigen into theorganism (e.g., a mammal) by any suitable manner. In some embodiments,the Myc peptide is a fusion peptide. In some embodiments, the Mycpeptide is a TAT-Myc peptide as described herein. In some embodiments,the organism is a xenomouse. In certain embodiments, the organism (e.g.,a mammal) further comprises a nucleic acid sequence (e.g., transgenicnucleic acid sequence) encoding the selected antigen. In someembodiments, the nucleic acid sequence (e.g., transgenic nucleic acidsequence) encoding the selected antigen comprises a B-cell-selectivepromoter operably linked to the open reading frame encoding the selectedantigen.

In some embodiments, a method described herein further comprisesrecovering from the organism (e.g., a mammal) a cell (e.g., B-cells)that express the antibody that specifically binds the selected antigen.In certain embodiments, a method described herein further comprisesrecovering from the cells (e.g., mammalian cells), B-cells) the antibodythat specifically binds the selected antigen. In some embodiments, amethod described herein further comprises recovering from the organism(e.g., a mammal) the antibody that specifically binds the selectedantigen.

In some embodiments, provided herein is a method for producing anantibody that specifically binds to an antigen, comprising: contacting acell with a selected antigen, wherein the cell (e.g., a mammalian cell)over-expresses an onco-peptide (e.g., recombinant onco-peptide; e.g.,Myc). In some embodiments, the Myc peptide is a fusion peptide. In someembodiments, the Myc peptide is a TAT-Myc peptide as described herein.

In some embodiments, the onco-peptide is a peptide that promotes cellsurvival and/or proliferation. In certain embodiments, the cell is amammalian cell. In some embodiments, the cell is a human cell. In someembodiments, the cell is a B-cell. In some embodiments, the cell is ahematopoietic cell. In some embodiments, the cell is a human B-cell. Insome embodiments, the cell is a human hematopoietic cell. In someembodiments, the cell (e.g., a mammalian cell) expresses CD79 on itscell surface. In some embodiments, the cell (e.g., a mammalian cell)comprising a nucleic acid sequence (e.g., a transgenic sequence)comprises an intact salvage pathway for purine biosynthesis. In someembodiments, the cell (e.g., a mammalian cell) comprising a nucleic acidsequence (e.g., a transgenic sequence) comprises an intact salvagepathway for purine biosynthesis and is tolerant and or anergic to theselected antigen. In some embodiments, the cell (e.g., a mammalian cell)is a B-cell, a B-cell progenitor or precursor, or a hematopoietic stemcell. In certain embodiments, the antibody specifically binds to theselected antigen. In certain embodiments, the onco-peptide is a peptidethat promotes cell survival and/or proliferation.

In some embodiments, over-expression of MYC is induced by contacting thecell with a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In certaininstances, the down-regulation of an Max-1 gene and/or polypeptideupregulates the expression of a MYC proto-oncogene and/or a polypeptideencoded by a MYC proto-oncogene. In some embodiments, the small moleculeis an antagonist of Mxi-1. In certain instances, the down-regulation ofan Mxi-1 gene and/or polypeptide upregulates the expression of a MYCproto-oncogene and/or a polypeptide encoded by a MYC proto-oncogene. Insome embodiments, the small molecule is an antagonist of MAD. In certaininstances, the down-regulation of a MAD-1 gene and/or polypeptideupregulates the expression of a MYC proto-oncogene and/or a polypeptideencoded by a MYC proto-oncogene. In some embodiments, over-expression ofMYC is induced by contacting the cell with a siRNA molecule for Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments,over-expression of MYC is induced by contacting the cell with a shRNAmolecule for Max-1, Mxi-1, MAD, or a combination thereof.

In certain embodiments, the cell (e.g., a mammalian cell) is contactedwith the selected antigen by any suitable method. In specificembodiments, the cell (e.g., a mammalian cell) comprises a nucleic acidsequence (e.g., a transgenic sequence) that encodes a nucleic acidsequence (e.g., a transgenic sequence) that encodes the selectedantigen. In some embodiments, the selected antigen is a self-antigen.

In certain embodiments, wherein a B-cell precursor or progenitor or ahematopoietic stem cell are used, a method described herein furthercomprises allowing or inducing differentiation of the cell (e.g., amammalian cell) to a B-cell.

In some embodiments, a MYC sequence (e.g., a transgenic sequence)further comprises a B-cell selective promoter or an inducible promoter.In some embodiments, a nucleic acid sequence (e.g., a transgenicsequence) encoding the selected antigen further comprises a B-cellselective promoter or an inducible promoter. In some embodiments, aB-cell selective promoter is, by way of non-limiting example, the Eμpromoter. In some embodiments, the inducible promoter comprises, by wayof non-limiting example, one or more TREs. In certain embodiments, thecell (e.g., a mammalian cell) further comprises a nucleic acid sequence(e.g., a transgenic sequence) that encodes a tTA peptide or an rtTApeptide. In some embodiments, the nucleic acid sequence (e.g.,transgenic nucleic acid sequence) encoding the tTA peptide or the rtTApeptide comprises a B-cell-selective promoter operably linked to theopen reading frame encoding the tTA peptide or the rtTA peptide. In someembodiments, the nucleic acid sequence (e.g., transgenic nucleic acidsequence) encoding the tTA peptide or the rtTA peptide comprises theMMTV promoter operably linked to the open reading frame encoding the tTApeptide or the rtTA peptide. Furthermore, in some embodiments, whereinan inducible promoter comprises one or more TREs, a method describedherein further comprises contacting the cell (e.g., a mammalian cell)with doxycycline, tetracycline, or an analog thereof.

In some embodiments, the onco-peptide is a fusion peptide that comprisesa receptor that activates the onco-peptide activity (e.g., cell survivaland/or proliferation) when bound with a ligand. In some embodiments, thereceptor is an estrogen receptor (ER). In specific embodiments, theonco-peptide is a Myc-ER fusion peptide. In some embodiments, thereceptor is an clucocorticoid receptor (GR). In specific embodiments,the onco-peptide is a Myc-GR fusion peptide. In certain embodiments, amethod described herein further comprises contacting a cell comprising afusion peptide comprising a receptor with a ligand that specificallybinds to the receptor. In specific embodiments, a method describedherein further comprises contacting a cell comprising a Myc-ER peptidewith an estrogen receptor modulator (e.g., an ER agonist or antagonist).In specific embodiments, a method described herein further comprisescontacting a cell comprising a Myc-GR peptide with an glucocorticoidreceptor modulator (e.g., a GR agonist or antagonist).

In some embodiments, the onco-peptide is a fusion peptide that comprisesa (a) a transporter peptide sequence (e.g., TAT); and (b) a MYC sequence(e.g., c-MYC). In some embodiments, the fusion peptide is a peptide ofFormula (I):

transporter peptide sequence-MYC sequence.

In some embodiments, a fusion peptide disclosed herein comprises (a) atransporter peptide sequence; (b) a MYC sequence; and (c) one or moremolecules that link the transporter peptide sequence and the MYCsequence (i.e., “X”). In some embodiments, the fusion peptide is apeptide of Formula (II):

transporter peptide sequence-X-MYC sequence,

wherein -X- is molecule that links the transporter peptide sequence andthe MYC sequence. In some embodiments, -X- is an amino acid. In someembodiments, -X- is at least one amino acid.

In certain embodiments, a method described herein further comprisesinducing MYC expression, Myc activity, or a combination thereof, in thecell (e.g., a mammalian cell). In some embodiments, a method describedherein further comprises proliferating or inducing proliferation of thecell (e.g., a mammalian cell). In certain embodiments, the cells (e.g.,mammalian cells) are proliferated at least until forming a lymphoma(e.g., a B-cell lymphoma). In some embodiments, the proliferating cellsare lymphoma cells. In certain embodiments, the cells (e.g., mammaliancells) do not require and/or are not fused, e.g., with a myeloma, priorto proliferation (e.g., in order to provide a cell population sufficientto produce a significant or therapeutic amount of the antibody in areasonable period of time).

In some embodiments, the method further comprises recovering theantibody that specifically binds the selected antigen. In someembodiments, the antibody produced is soluble and is not membrane bound.In some embodiments, the antibody produced is membrane bound. In someembodiments, the antibody produced is intracellular.

In some embodiments, the cell (e.g., a mammalian cell) is present in anorganism (e.g., a mammal). In some embodiments, the organism is axenomouse. In certain embodiments, the organism (e.g., a mammal)comprises a nucleic acid sequence (e.g., a transgenic sequence) thatencodes an onco-peptide. In some embodiments, the organism (e.g., amammal) further comprises a nucleic acid sequence (e.g., a transgenicsequence) that encodes a tTA peptide or an rtTA peptide. In someembodiments, the nucleic acid sequence (e.g., transgenic nucleic acidsequence) encoding the tTA peptide or the rtTA peptide comprises aB-cell-selective promoter operably linked to the open reading frameencoding the tTA peptide or the rtTA peptide. In some embodiments, thenucleic acid sequence (e.g., transgenic nucleic acid sequence) encodingthe tTA peptide or the rtTA peptide comprises the MMTV promoter operablylinked to the open reading frame encoding the tTA peptide or the rtTApeptide.

In certain embodiments, contacting the cell (e.g., a mammalian cell)comprising MYC (e.g., a transgenic MYC) comprises introducing (orotherwise providing to) the selected antigen into the organism (e.g., amammal) by any suitable manner. In certain embodiments, the organism(e.g., a mammal) further comprises a nucleic acid sequence (e.g., atransgenic sequence) encoding the selected antigen. In some embodiments,the nucleic acid sequence (e.g., transgenic nucleic acid sequence)encoding the selected antigen comprises a B-cell-selective promoteroperably linked to the open reading frame encoding the selected antigen.

In some embodiments, a method described herein further comprisesproviding doxycycline, tetracycline, or an analog thereof to theorganism (e.g., a mammal) to suppress tTA-dependent expression of thenucleic acid sequence (e.g., transgenic nucleic acid sequence) encodinga Myc peptide (e.g., a recombinant Myc peptide (e.g., a Myc fusionpeptide as described herein)). In some embodiments, the onco-peptide isa fusion peptide. In some embodiments, the onco-peptide is a fusionpeptide comprising a PTD. In some embodiments, the onco-peptide isTAT-MYC. In some embodiments, the onco-peptide is Myc-ER. In someembodiments, the onco-peptide is Myc-GR. In some embodiments, a methoddescribed herein further comprises (a) providing doxycycline,tetracycline, or an analog thereof to the organism (e.g., a mammal) fora period of time sufficient to suppress tTA-dependent expression of thenucleic acid sequence (e.g., transgenic nucleic acid sequence) encodingthe Myc peptide, and (b) withdrawing the doxycycline, tetracycline, oranalog thereof after the period of time to induce tTA-dependentexpression of the nucleic acid sequence (e.g., transgenic nucleic acidsequence) encoding the Myc peptide.

In some embodiments, a method described herein further comprisesrecovering from the organism (e.g., a mammal) a cell (e.g., B-cell) thatexpress the antibody that specifically binds the selected antigen. Incertain embodiments, a method described herein further comprisesrecovering from the cell (e.g., a mammalian cell) (e.g., B-cell) theantibody that specifically binds the selected antigen. In someembodiments, a method described herein further comprises recovering fromthe organism (e.g., a mammal) the antibody that specifically binds theselected antigen.

In specific embodiments, provided herein is a method of producing anantibody that specifically binds to an antigen, comprising: introducing(or otherwise providing to) a selected antigen into an organism (e.g., amammal), wherein the organism (e.g., a mammal) comprises a nucleic acidsequence (e.g., a transgenic sequence) that encodes an onco-peptide(e.g., recombinant onco-peptide; e.g., Myc), and comprises an induciblepromoter or a B-cell-selective promoter operably linked to the openreading frame of the encoded onco-peptide.

In some embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide is a fusion peptide comprising a PTD. Insome embodiments, the onco-peptide is TAT-MYC. In some embodiments, theonco-peptide is Myc-ER. In some embodiments, the onco-peptide is Myc-GR.

In some embodiments, the organism is a xenomouse.

In some embodiments, provided herein is a method of producing anantibody that specifically binds to an antigen, comprising:

-   -   a. providing a cell (e.g., a B-cell) expressing an antibody that        specifically binds an antigen, wherein the cell (e.g., a        mammalian cell) comprises an onco-peptide (e.g., a recombinant        onco-peptide; e.g., Myc) or a nucleic acid sequence (e.g., a        transgenic sequence) encoding an onco-peptide (e.g., a        recombinant onco-peptide; e.g., Myc); and    -   b. inducing activity of the onco-peptide in the cell (e.g., a        mammalian cell).

In some embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide is a fusion peptide comprising a PTD. Insome embodiments, the onco-peptide is TAT-MYC. In some embodiments, theonco-peptide is Myc-ER. In some embodiments, the onco-peptide is Myc-GR.

In some embodiments, the cell (e.g., a mammalian cell) is prepared bycontacting a cell with the selected antigen. In some embodiments, theselected antigen is a self-antigen. In certain embodiments, the antibodyspecifically binds to the selected antigen.

In some embodiments, the cell is a human cell. In certain embodiments,the cell (e.g., a mammalian cell) is a B-cell, a B-cell precursor orprogenitor, or a hematopoietic stem cell. In some embodiments, theB-cell is an anergic B-cell. In some embodiments, the cell (e.g., amammalian cell) expresses CD79 on its cell surface. In some embodiments,the B-cell is a mouse B-cell. In some embodiments, the cell (e.g., amammalian cell; e.g., B-cell) is from an organism (e.g., a mammal; e.g.,a xenomouse) that was administered (i.e., inoculated with, or immunizedagainst) the selected antigen. In some embodiments, the organism is axenomouse. In some embodiments, the organism (e.g., a mammal) comprisesan onco-peptide (e.g., a recombinant onco-peptide; e.g., Myc) or anucleic acid sequence (e.g., a transgenic sequence) encoding anonco-peptide (e.g., a recombinant onco-peptide; e.g., Myc). In someembodiments, the onco-peptide is a fusion peptide. In some embodiments,the onco-peptide is a fusion peptide comprising a PTD. In someembodiments, the onco-peptide is TAT-MYC. In some embodiments, theonco-peptide is Myc-ER. In some embodiments, the onco-peptide is Myc-GR.In some embodiments, the organism (e.g., a mammal) further comprises anucleic acid sequence (e.g., a transgenic sequence) that encodes theselected antigen. In some embodiments, a MYC sequence (e.g., atransgenic sequence) further comprises a B-cell selective promoter or aninducible promoter. In some embodiments, a nucleic acid sequence (e.g.,a transgenic sequence) encoding the selected antigen further comprises aB-cell selective promoter or an inducible promoter. In some embodiments,a B-cell selective promoter is, by way of non-limiting example, the Eμpromoter. In some embodiments, the inducible promoter comprises, by wayof non-limiting example, one or more TREs.

In certain embodiments, wherein a B-cell precursor or progenitor or ahematopoietic stem cell are used, a method described herein furthercomprises allowing or inducing differentiation of the cell (e.g., amammalian cell) into a B-cell.

In some embodiments, the method further comprises recovering theantibody. In certain embodiments, activity of the onco-peptide inducesexpansion of the cell (e.g., a mammalian cell) population.

In some embodiments, the cell (e.g., a mammalian cell) (e.g., B-cell) ispresent in an organism (e.g., a mammal) and inducing the activity of theonco-peptide (e.g., recombinant onco-peptide; e.g., Myc) in the cell(e.g., a mammalian cell) (e.g., B-cell) occurs in vivo. In someembodiments, the organism is a xenomouse. In some embodiments, themethod further comprises introducing (or otherwise providing to) anonco-peptide (e.g., a recombinant onco-peptide; e.g., Myc) or thenucleic acid sequence (e.g., transgenic nucleic acid sequence) encodingan onco-peptide (e.g., a recombinant onco-peptide; e.g., Myc) into thecell (e.g., a mammalian cell). In some embodiments, the onco-peptide orthe nucleic acid sequence (e.g., transgenic nucleic acid sequence)encoding the onco-peptide is introduced into the cell (e.g., a mammaliancell) ex vivo and then the cell (e.g., a mammalian cell) is introducedinto an organism (e.g., a mammal). In some embodiments, the organism isa xenomouse. In specific embodiments, the process comprises introducing(or otherwise providing to) a onco-peptide (e.g., recombinantonco-peptide; e.g., Myc) into a cell (e.g., B-cell) ex vivo. In someembodiments, the onco-peptide (e.g., recombinant onco-peptide; e.g.,Myc) comprises a protein transduction domain, e.g., HIV-1 Tat or Vpr(e.g., Tat-Myc or Vpr-Myc).

In some embodiments, inducing the activity of an onco-peptide includesinducing the expression of a nucleic acid sequence encoding theonco-peptide, inducing the activity of the onco-peptide, or acombination thereof. In certain instances, inducing activity of theonco-peptide includes inducing over-expression of the onco-peptide. Insome embodiments, inducible expression or over-expression of theonco-peptide is achieved by activating an inducible promoter of anucleic acid sequence (e.g., a transgenic sequence) that inducesexpression of an onco-peptide. In some embodiments, over-expression ofMYC is induced by contacting the cell with a small molecule, a biologic,a peptide, an antibody, or a combination thereof. In some embodiments,the small molecule is an antagonist of Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1. In some embodiments, the small molecule is anantagonist of Mxi-1. In some embodiments, the small molecule is anantagonist of MAD. In some embodiments, over-expression of MYC isinduced by contacting the cell with a siRNA molecule for Max-1, Mxi-1,MAD, or a combination thereof. In some embodiments, over-expression ofMYC is induced by contacting the cell with a shRNA molecule for Max-1,Mxi-1, MAD, or a combination thereof.

In certain embodiments, the nucleic acid sequence (e.g., transgenicnucleic acid sequence) encoding an onco-peptide (e.g., recombinantonco-peptide; e.g., Myc) comprises a promoter, e.g., a B-cell selectivepromoter or an inducible promoter, operably linked to the open readingframe encoding the onco-peptide (e.g., recombinant onco-peptide; e.g.,Myc). In some specific embodiments, the nucleic acid sequence (e.g.,transgenic nucleic acid sequence) encoding an onco-peptide (e.g.,recombinant onco-peptide; e.g., Myc) comprises a B-cell-selectivepromoter operably linked to an open reading frame. In certain specificembodiments, the nucleic acid sequence (e.g., transgenic nucleic acidsequence) encoding the onco-peptide comprises an inducible promoteroperably linked to an open reading frame for the Myc peptide. In someembodiments, the inducible promoter comprises one or more TREs and thecell (e.g., a mammalian cell) further expresses a tTA peptide or an rtTApeptide. In some embodiments, the cell (e.g., a mammalian cell)expresses the tTA peptide. In some embodiments, the onco-peptide is afusion peptide. In some embodiments, the onco-peptide is a fusionpeptide comprising a PTD. In some embodiments, the onco-peptide isTAT-MYC. In some embodiments, the onco-peptide is Myc-ER. In someembodiments, the onco-peptide is Myc-GR.

In some embodiments, the onco-peptide is a fusion peptide that isinducibly activated. In specific embodiments, the onco-peptide is afusion peptide comprising a receptor that activates the survival and/orproliferative characteristic of the onco-peptide when modulated (i.e.,bound with a ligand, such, as an agonist or antagonist). In specificembodiments, the onco-peptide (e.g., a recombinant onco-peptide; e.g.,Myc) is a fusion peptide comprising an estrogen receptor (e.g., Myc-ER).In some embodiments, inducing activity of the onco-peptide in the cell(e.g., a mammalian cell) comprises contacting the cell (e.g., amammalian cell) with an ER ligand. In specific embodiments, theonco-peptide (e.g., a recombinant onco-peptide; e.g., Myc) is a fusionpeptide comprising an glucocorticoid receptor (e.g., Myc-GR). In someembodiments, inducing activity of the onco-peptide in the cell (e.g., amammalian cell) comprises contacting the cell (e.g., a mammalian cell)with a GR ligand.

In some embodiments, the onco-peptide is a fusion peptide that comprisesa (a) a transporter peptide sequence (e.g., TAT); and (b) a MYC sequence(e.g., c-MYC). In some embodiments, the fusion peptide is a peptide ofFormula (I):

transporter peptide sequence-MYC sequence.

In some embodiments, a fusion peptide disclosed herein comprises (a) atransporter peptide sequence; (b) a MYC sequence; and (c) one or moremolecules that link the transporter peptide sequence and the MYCsequence (i.e., “X”). In some embodiments, the fusion peptide is apeptide of Formula (II):

transporter peptide sequence-X-MYC sequence,

wherein -X- is molecule that links the transporter peptide sequence andthe MYC sequence. In some embodiments, -X- is an amino acid. In someembodiments, -X- is at least one amino acid.

In some embodiments, the cell (e.g., a mammalian cell; e.g., a B-cell)comprising an onco-peptide (e.g., recombinant onco-peptide; e.g., Myc)or a nucleic acid sequence (e.g., a transgenic sequence) encoding anonco-peptide (e.g., a recombinant onco-peptide; e.g., Myc) is present inan organism (e.g., a mammal). In some embodiments, the Myc peptide is afusion peptide. In some embodiments, the Myc peptide is a TAT-Mycpeptide as described herein. In some embodiments, the organism is axenomouse. In some embodiments, the organism is administered (i.e.,immunized against, inoculated with) an antigen and allowed to mount animmune response to the selected antigen. In some embodiments, theorganism is exposed to the selected antigen by any suitable manner. Insome embodiments, the selected antigen is administered with an adjuvant.In some embodiments, the selected antigen is covalently linked to acarrier. Examples of carries include but are not limited to bovine serumalbumin, keyhole limpet hemocyanin, ovalbumin and hyroglobulin.

In some embodiments, a cell or organism utilized in a method disclosedherein includes those that inducibly over-express an onco-peptide (e.g.,recombinant onco-peptide; e.g., Myc) and are engineered to express anantigen. Such cells and organisms are prepared by any suitable methodincluding, by way of non-limiting example, retroviral mediatedtransduction of bone marrow hematopoietic stem cells, production oftransgenic organisms (or crossing the onco-peptide over-expressingorganisms with an organism that expresses the selected antigen), or anyother method for gene delivery into the cell (e.g., a mammalian cell) ororganism. In some embodiments, over-expression of MYC is induced bycontacting the cell with a small molecule, a biologic, a peptide, anantibody, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, over-expression of MYC is induced bycontacting the cell with a shRNA molecule for Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the cell (e.g., a mammaliancell) or organism is maintained under conditions in which anonco-peptide (e.g., recombinant onco-peptide; e.g., Myc) is notover-expressed until the production of antibodies is desired. In someembodiments, the cell (e.g., a mammalian cell) or organism is maintainedunder conditions in which the onco-peptide activity is not induced untilthe production of antibodies is desired. In some embodiments, the cell(e.g., a mammalian cell) or organism is maintained under conditionsunder which the onco-peptide is over-expressed in a cell (e.g., amammalian cell; e.g., a B-cell) that produces antibodies thatspecifically binds to the selected antigen.

In some embodiments, B cells specific for a heterologous antigen, a selfantigen, or an auto antigen are tolerant to the heterologous antigen,self antigen, or auto antigen. In some embodiments, induction ofonco-peptide (e.g., recombinant onco-peptide; e.g., Myc) activity intolerant B cells induces a break in tolerance to an antigen and inducesproduction of antibodies that specifically binds to the selectedantigen. In some embodiments, a tolerant B-cell is anergic to anantigen. In some embodiments, a tolerant B-cell is tolerant to anantigen that is immunologically similar to an auto antigen or selfantigen. In some embodiments, a tolerant B-cell is tolerant to anantigen that is a heterologous antigen. In some embodiments, theheterologous antigen is homologous to an auto antigen or self antigen.In some embodiments, the selected antigen specific B-cells produced byover-expression of MYC are removed from the organism and are cloned toproduce immortal antibody producing B-cells without the need for cellfusion to a myeloma partner. In some embodiments, the Myc peptide is afusion peptide. In some embodiments, the Myc peptide is a TAT-Mycpeptide as described herein.

In specific embodiments, provided herein is a method for producing anantibody that specifically binds to an antigen, comprising:

-   -   a. providing a B-cell that is tolerant to an antigen; and    -   b. inducing onco-peptide (e.g., recombinant onco-peptide; e.g.,        Myc) activity in a B-cell that is tolerant to the selected        antigen.        In some embodiments, the onco-peptide is a fusion peptide. In        some embodiments, the onco-peptide is a fusion peptide        comprising a PTD. In some embodiments, the onco-peptide is        TAT-MYC. In some embodiments, the onco-peptide is Myc-ER. In        some embodiments, the onco-peptide is Myc-GR. In certain        embodiments, induction of onco-peptide activity induces an        expansion of the B-cell population. In some embodiments, the        method further comprises recovering the antibody from a        plurality of recombinant B-cells generated by the expansion of        the B-cell.

In specific embodiments, provided herein is a method of producing anantibody that specifically binds to an antigen, comprising:administering to an immuno-deficient organism (e.g., a mammal) aplurality of hematopoietic stem cells (e.g., recombinant hematopoieticstem cells) that comprise a nucleic acid sequence (e.g., a transgenicsequence) encoding an onco-peptide (e.g., a recombinant onco-peptide;e.g., Myc); and contacting a selected antigen with the immuno-deficientorganism (e.g., a mammal). In some embodiments, the onco-peptide is afusion peptide. In some embodiments, the onco-peptide is a fusionpeptide comprising a PTD. In some embodiments, the onco-peptide isTAT-MYC. In some embodiments, the onco-peptide is Myc-ER. In someembodiments, the onco-peptide is Myc-GR.

In some embodiments, the organism is a xenomouse.

In certain embodiments, the onco-peptide is a Myc peptide. In someembodiments, the onco-peptide is a fusion peptide. In some embodiments,the onco-peptide is a fusion peptide comprising a PTD. In someembodiments, the onco-peptide is TAT-MYC. In some embodiments, theonco-peptide is inducibly activated (e.g., inducibly expressed or thefunction is inducibly activated). In certain embodiments, theonco-peptide is a fusion peptide comprising a receptor, and the functionof the onco-peptide is inducibly activated by modulating or binding thereceptor (e.g., with a ligand, such as an agonist or antagonist). Inspecific embodiments, the onco-peptide is Myc-ER. In specificembodiments, the onco-peptide is Myc-GR. In further embodiments, themethod further comprises inducing onco-peptide (e.g., recombinantonco-peptide; e.g., Myc) activity in the organism (e.g., a mammal). Insome embodiments, the organism is a xenomouse. For example, in the caseof Myc-ER, Myc activity is optionally induced by administering anestrogen receptor ligand to the immuno-deficient organism (e.g., amammal); in the case of a MYC nucleic acid sequence with an induciblepromoter comprising one or more TREs, Myc activity is induced byproviding doxycycline, tetracycline, or an analog thereof to theimmuno-deficient organism (e.g., a mammal); or in the case of Myc-GR,Myc activity is induced by administering a glucocorticoid receptorligand to the immune-deficient organism.

In some embodiments, the hematopoietic stem cells further comprise anucleic acid sequence (e.g., a transgenic sequence) encoding apolypeptide that inhibits apoptosis (e.g., Bcl-2, Bcl-x, Mcl-1).

In some embodiments, a method described herein further comprisesinducing a plurality of the hematopoietic stem cells to differentiateinto B-cells. In some embodiments, a method disclosed herein furthercomprises recovering a plurality of the B-cells that express an antibodythat specifically binds to the selected antigen from theimmuno-deficient organism (e.g., a mammal). In some embodiments, amethod described herein further comprises recovering the antibody fromthe immuno-deficient organism (e.g., a mammal) or from a plurality ofB-cells that express the antibody.

In some embodiments, the nucleic acid sequence (e.g., transgenic nucleicacid sequence) encoding an onco-peptide comprises an inducible promoteror a B-cell-selective promoter. In some embodiments, the nucleic acidsequence (e.g., transgenic nucleic acid sequence) encoding anonco-peptide comprises an inducible promoter comprising one or moreTREs. In some embodiments, the hematopoietic stem cells express tTA orrtTA. In some embodiments, a method described herein further comprisesproviding doxycycline, tetracycline, or an analog thereof to theimmuno-deficient organism (e.g., a mammal) to suppress the tTA-dependenttransactivation, and withdrawing the doxycycline, tetracycline, oranalog thereof to induce tTA-dependent transactivation. In someembodiments, the B-cell selective promoter is the Eμ promoter.

In some embodiments, an immuno-deficient organism (e.g., a mammal) isobtained by irradiating the organism (e.g., a mammal). In someembodiments, the immuno-deficient organism (e.g., a mammal) is aRag-1ko, Rag-2, SCID, DNA-PK, Ku70, Ku80, XRCC4, or μMT mouse. In someembodiments, the organism is a xenomouse.

In certain embodiments, the selected antigen is administered to theorganism by any suitable method. In some embodiments, the selectedantigen is a self antigen. In some embodiments, the organism is animmuno-deficient mammal. In some embodiments, the organism is animmuno-deficient mouse. In some embodiments, the organism is animmune-deficient xenomouse. In some embodiments, the immuno-deficientorganism (e.g., a mammal) expresses the selected antigen. In someembodiments, the immuno-deficient organism (e.g., a mammal) thatexpresses the selected antigen comprises an exogenous nucleic acidsequence that encodes the selected antigen. In some embodiments, theselected antigen is introduced by transfection with a nucleic acidexpression vector or infection with a recombinant virus expressionvector. In some embodiments, the recombinant virus expression vector isa recombinant lentivirus. In certain embodiments, the antibodyspecifically binds to the selected antigen. In some embodiments, the Mycpeptide is a fusion peptide. In some embodiments, the Myc peptide is aTAT-Myc peptide as described herein.

In some embodiments, hematopoietic stem cells that over-express MYC areused to reconstitute the peripheral lymphoid compartments ofimmuno-deficient organisms. In some embodiments, over-expression of MYCis induced by contacting the cell with a small molecule, a biologic, apeptide, an antibody, or a combination thereof. In some embodiments, thesmall molecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, over-expression of MYC is induced bycontacting the cell with a shRNA molecule for Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the immuno-deficient organismsare rendered immuno-deficient by lethal irradiation. In someembodiments, immuno-deficient organisms are lethally irradiated mice. Insome embodiments, the immuno-deficient organism is any immuno-deficientor immuno-compromised mouse. In some embodiments, the immuno-deficientanimal is a Rag-1 knock out, Rag-2, SCID, DNA-PK, Ku70, Ku80, XRCC4, orμMT mouse. The peripheral lymphoid compartment of an immuno-deficientanimal is typically reconstituted in 8-12 weeks after transplantation ofhematopoietic stem cells. In some embodiments, organisms that arereconstituted with hematopoietic stem cells that over-express MYC, arefurther genetically altered to express the selected antigen. In someembodiments, organisms that are reconstituted with hematopoietic stemcells that over-express MYC are administered (i.e., immunized with orinoculated against) the selected antigen.

In some embodiment B-cell populations (e.g., anergic B-cells, such asAN1/T3) are isolated from organisms reconstituted with hematopoieticstem cells and are used in any method described herein. In someembodiments, hematopoietic stem cells that over-express MYC reconstitutethe peripheral hematopoietic compartment of immuno-deficient organisms,are exposed to the selected antigen and develop into antigen specificAN1/T3 cell populations. In some embodiments, induction of Myc activityin these cells generates immortal antigen specific B-cells.

The hematopoietic stem cells utilized herein are prepared by anysuitable method. In some embodiments, hematopoietic stem cells areisolated from an organism that over-expresses Myc. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments, theorganism is a xenomouse. In some embodiments, hematopoietic stem cellsare isolated from organisms that comprise antigen specific B-cellpopulations. In some embodiments, hematopoietic stem cells are isolatedfrom organisms that comprise antigen binding B-cell populations. In someembodiments, hematopoietic stem cells are isolated from organisms thatcomprise antigen specific AN1/T3 cell populations. In some embodiments,hematopoietic stem cells are isolated from organisms that compriseantigen specific anergic B-cell populations. In some embodiments,hematopoietic stem cells are isolated from wild type organisms. In someembodiments, the organisms are mice (e.g., transgenic mice orgenetically altered mice). In some embodiments, organisms are treatedwith 5FU, in order to enrich for long-term hematopoietic stem cells, andinduce their proliferation in vivo. In some embodiments, the organisms(e.g., mice) are treated with from 0.01 to 100 mg/mouse of 5FU. In someembodiments, the organisms (e.g., mice) are treated with from 0.1 to 50mg/mouse of 5FU. In some embodiments, the organisms (e.g., mice) aretreated with from 1 to 10 mg/mouse of 5FU. In some embodiments, theorganisms (e.g., mice) are treated with 5 mg/mouse of 5FU. In someembodiments, bone marrow cells containing 5FU enriched populations ofhematopoietic stem cells are isolated, by any suitable method, from thefemurs and tibia bones of organisms that over-express MYC. In someembodiments, over-expression of MYC is induced by contacting the cellwith a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.In some embodiments, hematopoietic stem cells are isolated approximately1-10 days after administration of 5FU. In some embodiments,hematopoietic stem cells are isolated approximately 5 days afteradministration of 5FU. In some embodiments, hematopoietic stem cells arecultured in vitro in a media comprising human IL-3, IL-6 and Stem CellFactor (SCF). In certain embodiments, isolated hematopoietic stem cellsover-express MYC. In some embodiments, over-expression of MYC is inducedby contacting the cell with a small molecule, a biologic, a peptide, anantibody, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, hematopoietic stem cells are geneticallyaltered to over-express MYC in vitro by introducing (or otherwiseproviding to) a nucleic acid into the hematopoietic stem cells in whichthe nucleic acid directs the over-expression of MYC. In someembodiments, the over-expression of MYC is inducible. In someembodiments, the hematopoietic stem cells are genetically altered toover-express MYC by infecting the hematopoietic stem cells in vitro witha virus that directs the over-expression of MYC. In some embodiments,the virus is a lentivirus, retrovirus or adenovirus. In someembodiments, the hematopoietic stem cells are genetically altered toover-express MYC by transfection with a nucleic acid. In someembodiments, the hematopoietic stem cells are also genetically alteredto express a reporter gene. In certain embodiments, reporter genes areutilized and allow selection or isolation of cells that are geneticallyaltered to express Myc. In some embodiments, the reporter gene is GFP(green fluorescent protein). In some embodiments, hematopoietic stemcells are transduced with a virus that directs the over-expression ofMYC and also GFP. In some embodiments, the hematopoietic stem cells areinfected with a lentivirus that directs the over-expression of MYC andGFP. In some embodiments, provided herein are hematopoietic stem cellsthat over-express MYC, as described herein, and are used to generateimmortal antibody producing B-cells.

In various embodiments, of any of the methods disclosed herein,polyclonal populations of antibodies that specifically binds to theselected antigen, as well as the cell (e.g., a mammalian cell) producingsuch antibodies, are isolated directly from the tissues (spleen, lymphnodes, blood, etc.) or serum of organisms. In some embodiments,monoclonal populations of antibody producing cells are isolated fromorganisms, and monoclonal antibodies are recovered from culture media.

In some embodiments, provided herein is any isolated B-cell prepared bya method described herein. In some embodiments, provided herein is acell (e.g., a mammalian cell) that has been engineered to produce arecombinant form of any antibody prepared according any processdisclosed herein. In certain embodiments, provided herein is a B-cellthat expresses a human or humanized antibody, and an onco-peptide (e.g.,recombinant onco-peptide; e.g., Myc), a nucleic acid sequence (e.g., atransgenic sequence) that encodes an onco-peptide (e.g., recombinantonco-peptide; e.g., Myc), and/or wherein the B-cell over-expresses Myc.In some embodiments, over-expression of MYC is induced by contacting thecell with a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.In some embodiments, over-expression of MYC is induced by contacting thecell with a shRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In certain embodiments, the onco-peptide is a fusion peptidecomprising a transduction domain (e.g., Tat-Myc or Vpr-Myc), a receptorthat activates function of the onco-peptide when modulated or bound(e.g., Myc-ER, Myc-GR), or a combination thereof (e.g., Tat-Myc-ER,Tat-Myc-GR, Vpr-Myc-ER, or Vpr-Myc-GR). In some embodiments, providedherein is an isolated antibody prepared according to the process of anyof the methods disclosed herein.

Human Antibodies

In certain embodiments, a method disclosed herein is utilized to preparehuman or humanized antibodies. In some embodiments, the cells (e.g.,mammalian cells) or organisms of any of a method disclosed hereinexpress human antibodies. In some embodiments, the organism is axenomouse.

In some embodiments, provided herein is a method of producing a human orhumanized antibody comprising: providing a cell (e.g., a mammalian cell)that expresses human antibodies and comprises a nucleic acid sequence(e.g., a transgenic sequence) that encodes a Myc peptide, or a Mycpeptide; and contacting the cell (e.g., a mammalian cell) with aselected antigen. In some embodiments, the onco-peptide is a fusionpeptide. In some embodiments, the onco-peptide is a fusion peptidecomprising a PTD. In some embodiments, the onco-peptide is TAT-MYC. Insome embodiments, the onco-peptide is Myc-ER. In some embodiments, theonco-peptide is Myc-GR.

In some embodiments, the cell (e.g., a mammalian cell) that expresseshuman antibodies and comprises a nucleic acid sequence (e.g., atransgenic sequence) that encodes the Myc peptide, or the Myc is a cellthat expresses cell-surface CD79. In some embodiments, the cell (e.g., amammalian cell) expresses CD79 and comprises an intact salvage pathwayfor purine biosynthesis. In some embodiments, the cell (e.g., amammalian cell) expresses CD79, comprises an intact salvage pathway forpurine biosynthesis and is tolerant and or anergic to the selectedantigen. In some embodiments, the cell (e.g., a mammalian cell) thatexpresses human antibodies and comprises the nucleic acid sequence(e.g., a transgenic sequence) that encodes a Myc peptide or a Mycpeptide is a B-cell, a B-cell progenitor or precursor, or ahematopoietic stem cell In some embodiments, the B-cell over-expressesMyc. In some embodiments, over-expression of MYC is induced bycontacting the cell with a small molecule, a biologic, a peptide, anantibody, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, over-expression of MYC is induced bycontacting the cell with a shRNA molecule for Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the cell (e.g., a mammaliancell) that expresses human antibodies and comprises the nucleic acidsequence that encodes a Myc peptide or the Myc peptide is present in anorganism (e.g., a mammal), and wherein the method further comprisesrecovering the antibody producing cell from the organism (e.g., amammal). In some embodiments, the Myc peptide is a fusion peptide. Insome embodiments, the Myc peptide is a TAT-Myc peptide as describedherein. In some embodiments, the organism is a xenomouse. In someembodiments, the organism (e.g., a mammal) is a mouse. In someembodiments, the organism (e.g., a mammal) is an MMTV-tTA/TRE-MYC mouse.In some embodiments, the organism (e.g., a mammal) is obtained by: (a)presenting an immuno-deficient organism (e.g., a mammal); and (b)administering to the organism (e.g., a mammal) a plurality ofhematopoietic stem cells that over-express MYC. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments,over-expression of MYC is induced by contacting the cell with a shRNAmolecule for Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the hematopoietic stem cells are human stem cells,including by way of example primary human cord blood. In furtherembodiments, the human stem cells are transduced to produceconditionally-activated MYC, including by way of example only MYC-ER andMyc-GR. In further or alternative embodiments, the aforementioned humanstem cells are also transduced with the cDNA of an antigen: in suchembodiments, the human stem cells will produce both the selected antigenand the conditionally-activated MYC. In further or alternativeembodiments, the resulting human stem cells are administered to animmuno-deficient organism (e.g., a mammal)—as one option, upon detectionof B-cells in the periphery, the immuno-deficient organism (e.g., amammal) is provided with the agent that induces conditional expressionof MYC. Alternatively, the human stem cells that have been transduced toproduce conditionally-activated Myc are provided directly to theimmuno-deficient organism (e.g., a mammal); upon detection of theB-cells in the periphery, the resulting organism (e.g., a mammal) isprovided with lymphoma cells (including lymphoma cells from the samespecies or genera as the immuno-deficient organism (e.g., a mammal))that express the selected antigen(s) of interest; the resulting organism(e.g., a mammal) is then provided with the agent thatconditionally-activates MYC.

In any of the embodiments, described herein, the immuno-deficientorganism (e.g., a mammal) is obtained by irradiating the organism (e.g.,a mammal). In some embodiments, the immuno-deficient organism (e.g., amammal) is a Rag-1ko, Rag-2, SCID, DNA-PK, Ku70, Ku80, XRCC4, or μMTmouse. In some embodiments, the organism (e.g., a mammal) expresses theselected antigen. In some embodiments, a method described herein furthercomprises recovering the antibody produced by the antibody producingcell. In some embodiments, a method described herein further comprisessubjecting the antibody producing cell to conditions that induceover-expression of MYC. In some embodiments, the nucleic acid sequence(e.g., a transgenic sequence) that encodes the Myc peptide comprises aninducible promoter or a B-cell-selective promoter. In some embodiments,prior to isolating the antibody producing cell from the organism (e.g.,a mammal) the antibody producing cell is subjected to conditions thatinduce over-expression of MYC. In some embodiments, over-expression ofMYC is induced by contacting the cell with a small molecule, a biologic,a peptide, an antibody, or a combination thereof. In some embodiments,the small molecule is an antagonist of Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1. In some embodiments, the small molecule is anantagonist of Mxi-1. In some embodiments, the small molecule is anantagonist of MAD. In some embodiments, over-expression of MYC isinduced by contacting the cell with a siRNA molecule for Max-1, Mxi-1,MAD, or a combination thereof. In some embodiments, over-expression ofMYC is induced by contacting the cell with a shRNA molecule for Max-1,Mxi-1, MAD, or a combination thereof.

In some embodiments, subsequent to isolating the antibody producing cellfrom the organism (e.g., a mammal) the antibody producing cell issubjected to conditions that induce over-expression of MYC. In someembodiments, over-expression of MYC is induced by contacting the cellwith a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.In some embodiments, over-expression of MYC is induced by contacting thecell with a shRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the organism (e.g., a mammal) is anMMTV-rtTA/TRE-MYC; MMTV-rtTA/TRE-MYC/Rag-1−/−; MMTV-tTA/TRE-MYC; orMMTV-tTA/TRE-MYC/Rag-1−/− mouse and the conditions that induceover-expression of MYC are exposure to doxycycline, tetracycline, or ananalog thereof. In some embodiments, the Myc peptide is a Myc-ER fusionpeptide. In some embodiments, a method described herein furthercomprises contacting the cell (e.g., a mammalian cell) with an estrogenreceptor ligand. In some embodiments, the cell (e.g., a mammalian cell)is contacted with the selected antigen in the absence of an estrogenreceptor ligand. In some embodiments, the Myc peptide is a Myc-GR fusionpeptide. In some embodiments, a method described herein furthercomprises contacting the cell (e.g., a mammalian cell) with aglucocorticoid receptor ligand. In some embodiments, the cell (e.g., amammalian cell) is contacted with the selected antigen in the absence ofan glucocorticoid receptor ligand.

In some embodiments, provided herein is a method of producing anantibody comprising: (a) contacting a cell comprising Myc (e.g.,recombinant Myc) with a selected antigen; (b) recovering the cell (e.g.,a mammalian cell) comprising Myc (e.g., recombinant Myc) wherein thecell (e.g., a mammalian cell) is now an immortal cell; and (c) isolatingthe antibody from the immortal cell wherein the antibody bindsspecifically to the selected antigen. In some embodiments, theonco-peptide is a fusion peptide. In some embodiments, the onco-peptideis a fusion peptide comprising a PTD. In some embodiments, theonco-peptide is TAT-MYC. In some embodiments, the onco-peptide isMyc-ER. In some embodiments, the onco-peptide is Myc-GR. In someembodiments, the antibody is human or humanized. In some embodiments,the antibody is CDR engrafted. In some embodiments, the antibody ischimeric. In some embodiments, the antibody is a human IgG. In someembodiments, the antibody is or comprises one or more polypeptidesderived from a human IgG1, IgG4, IgG2, IgD, IgA or IgM. In someembodiments, the cell (is a mammalian cell. In some embodiments, thecell (e.g., a mammalian cell) is a chicken B-cell. In some embodiments,the cell (e.g., a mammalian cell) is a mouse cell. In some embodiments,the cell (e.g., a mammalian cell) is present in an organism. In someembodiments, the organism is a xenomouse. In some embodiments, the cell(e.g., a mammalian cell) is an AN1/T3 cell. In some embodiments, thecell (e.g., a mammalian cell) is a HSC. In some embodiments, the cell(e.g., a mammalian cell) is an immature B-cell. In some embodiments, thecell (e.g., a mammalian cell) comprises a nucleic acid sequence (e.g., atransgenic sequence) encoding a Myc peptide (e.g., a recombinant Mycpeptide (e.g., a Myc fusion peptide as described herein)). In someembodiments, the Myc peptide is a fusion peptide. In some embodiments,the Myc peptide is a TAT-Myc peptide as described herein. In someembodiments, the cell (e.g., a mammalian cell) comprises one or morenucleic acid sequences (e.g., transgenic sequences) that encode a humanor humanized antibody. In some embodiments, the cell (e.g., a mammaliancell) comprises cell surface expression of CD79. In some embodiments,the cell (e.g., a mammalian cell) comprises a cell surface B-cellreceptor. In some embodiments, the cell (e.g., a mammalian cell) bindsspecifically to the selected antigen. In some embodiments, the cell(e.g., a mammalian cell) is anergic or tolerant to the selected antigen.In some embodiments, the cell (e.g., a mammalian cell) is anergic ortolerant to the selected antigen prior to contacting antigen in thepresence of Myc (e.g., recombinant Myc) activity. In some embodiments,the Myc peptide is a fusion peptide. In some embodiments, the Mycpeptide is a TAT-Myc peptide as described herein. In some embodiments,the cell (e.g., a mammalian cell) is mortal (i.e., not immortalized)and/or non-malignant. In some embodiments, the cell (e.g., a mammaliancell) is mortal (i.e., not immortalized) and/or non-malignant prior tocontacting antigen in the presence of Myc (e.g., recombinant Myc)activity. In some embodiments, the cell (e.g., a mammalian cell)comprises an intact, functional salvage pathway for purine biosynthesis.In some embodiment the cell (e.g., a mammalian cell) comprises afunctional hypoxanthine-guanine phosphoribosyltransferase enzyme. Insome embodiments, the immortal cell is malignant. In some embodiments,the immortal cell is a lymphoma. In some embodiments, the immortal cellexpresses CD79. In some embodiments, the immortal cell comprises Myc(e.g., recombinant Myc) activity. In some embodiments, the immortal cellcomprises an intact, functional salvage pathway for purine biosynthesis.In some embodiment the immortal cell comprises a functionalhypoxanthine-guanine phosphoribosyltransferase enzyme. In someembodiments, the immortal cell comprises no more than one nucleus. Insome embodiments, the immortal cell is not a hybridoma. In someembodiments, the immortal cell is not a myeloma. In some embodiments,the immortal cell comprises the same number of chromosomes as werepresent in the cell (e.g., a mammalian cell) Myc (e.g., recombinant Myc)activity prior to contacting the selected antigen. In some embodiments,the immortal cell comprises no more than 5 additional chromosomes thanwere present in the cell (e.g., a mammalian cell) Myc (e.g., recombinantMyc) activity prior to contacting the selected antigen. In someembodiments, the immortal cell comprises no more than 4 additionalchromosomes than were present in the cell (e.g., a mammalian cell) Myc(e.g., recombinant Myc) activity prior to contacting the selectedantigen. In some embodiments, the immortal cell comprises no more than 3additional chromosomes than were present in the cell (e.g., a mammaliancell) Myc (e.g., recombinant Myc) activity prior to contacting theselected antigen. In some embodiments, the immortal cell comprises nomore than 2 additional chromosomes than were present in the cell (e.g.,a mammalian cell) Myc (e.g., recombinant Myc) activity prior tocontacting the selected antigen. In some embodiments, the immortal cellcomprises no more than 1 additional chromosome than was present in thecell (e.g., a mammalian cell) Myc (e.g., recombinant Myc) activity priorto contacting the selected antigen. In some embodiments, the immortalcell comprises no additional chromosomes than were present in the cell(e.g., a mammalian cell) Myc (e.g., recombinant Myc) activity prior tocontacting the selected antigen. In some embodiments, Myc (e.g.,recombinant Myc) activity is inducible. In some embodiments, Myc (e.g.,recombinant Myc) activity is inducible and Myc (e.g., recombinant Myc)activity is not present in the cell (e.g., a mammalian cell) prior tocontact with antigen. In some embodiments, the cell (e.g., a mammaliancell) over-expresses Myc. In some embodiments, over-expression of MYC isinduced by contacting the cell with a small molecule, a biologic, apeptide, an antibody, or a combination thereof. In some embodiments, thesmall molecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, over-expression of MYC is induced bycontacting the cell with a shRNA molecule for Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the selected antigen is anauto antigen, a self antigen or a tolerant antigen. In some embodiments,the selected antigen is an auto antigen or a self antigen that is nativeto the organism. In some embodiments, the selected antigen is homologousto an auto antigen, self antigen or tolerant antigen. In someembodiments, the selected antigen is from 60-100% homologous. In someembodiments, the selected antigen is 60, 65, 70, 75, 80, 85, 90 or 95%homologous to a self antigen, auto antigen or tolerant antigen. In someembodiments, the selected antigen is 91, 92, 93, 94, 95, 96, 97, 98 or99% homologous to a self antigen, auto antigen or tolerant antigen. Insome embodiments, the selected antigen binds to a BCR but fails totransduce a signal through the BCR in the absence of Myc (e.g.,recombinant Myc) activity. In some embodiments, the selected antigenfails to induce an immune response when immunized into an organism inthe absence of Myc (e.g., recombinant Myc) activity. In someembodiments, contacting a cell comprising Myc (e.g., recombinant Myc)activity with the selected antigen comprises over-expression of theselected antigen in an organism by a nucleic acid sequence (e.g., atransgenic sequence) encoding the selected antigen. In some embodiments,contacting a cell comprising Myc (e.g., recombinant Myc) activity withthe selected antigen comprises over-expression of the selected antigenin the cell (e.g., a mammalian cell) by a nucleic acid sequence (e.g., atransgenic sequence) encoding the selected antigen. In some embodiments,contacting a cell comprising Myc (e.g., recombinant Myc) activity withthe selected antigen comprises immunizing an organism with the selectedantigen.

In some embodiments, a method described herein is used to produceimmortal cell lines that produce human antibodies. In some embodiments,organisms are produced that are genetically altered to produce humanantibodies and over-express MYC. In some embodiments, over-expression ofMYC is induced by contacting the cell with a small molecule, a biologic,a peptide, an antibody, or a combination thereof. In some embodiments,the small molecule is an antagonist of Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1. In some embodiments, the small molecule is anantagonist of Mxi-1. In some embodiments, the small molecule is anantagonist of MAD. In some embodiments, over-expression of MYC isinduced by contacting the cell with a siRNA molecule for Max-1, Mxi-1,MAD, or a combination thereof. In some embodiments, the organisms aremice (e.g., a xenomouse). In some embodiment the organisms are producedby cross breeding an organism that is genetically altered to produce ahuman antibody with an organism that over-expresses Myc. In someembodiments, over-expression of MYC is induced by contacting the cellwith a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.In some embodiments, the organism is genetically altered to produce ahuman antibody, a CDR engrafted antibody or a chimeric antibody. In someembodiments, a mouse strain that carries a transgenic BAC constructencoding the human immunoglobulin locus (IgH and IgL) is crossed with amouse that that over-expresses Myc. In some embodiments, over-expressionof MYC is induced by contacting the cell with a small molecule, abiologic, a peptide, an antibody, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1, Mxi-1, MAD,or a combination thereof. In some embodiments, the small molecule is anantagonist of Max-1. In some embodiments, the small molecule is anantagonist of Mxi-1. In some embodiments, the small molecule is anantagonist of MAD. In some embodiments, over-expression of MYC isinduced by contacting the cell with a siRNA molecule for Max-1, Mxi-1,MAD, or a combination thereof. In some embodiments, the mouse thatover-expresses Myc is an Eμ-MYC mouse or a MMTV-rtTA/TRE-MYC mouse. Insome embodiments, the mouse that over-expresses Myc expresses Myc-ER. Insome embodiments, the mouse that over-expresses Myc expresses Myc-GR. Insome embodiments, organisms that are genetically altered to producehuman antibodies and over-express MYC are exposed to an antigen. In someembodiments, organisms produced that are genetically altered to producehuman antibodies and over-express MYC are immunized with an antigen. Insome embodiments, organisms produced that are genetically altered toproduce human antibodies and over-express MYC also express a selfantigen, neo-self antigen, an auto antigen or an antigen. In someembodiments, hematopoietic stem cells are produced from the organismsdescribed herein that are genetically altered to produce humanantibodies and that over-express MYC. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments,hematopoietic stem cells produced from any animal described herein areused to generate immortal antibody producing B-cells. In someembodiments, immortal antibody producing B-cells are produced by themethod described herein using organisms that are genetically altered toproduce human antibodies and that over-express MYC. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments, theantibodies produce by the methods described herein are humanizedantibodies, CDR engrafted antibodies or chimeric antibodies.

In some embodiments, provided herein is a method of producing anantibody comprising: (a) contacting a cell comprising Myc (e.g.,recombinant Myc) activity with a selected antigen, wherein the cell(e.g., a mammalian cell) is present in an organism (e.g., a mammal), (b)recovering the cell (e.g., a mammalian cell) wherein the cell (e.g., amammalian cell) is now an immortal cell and (c) isolating the antibodyfrom the immortal cell wherein the antibody binds specifically to theselected antigen. In some embodiments, Myc is a fusion peptide. In someembodiments, Myc is a fusion peptide comprising a PTD. In someembodiments, Myc is TAT-MYC. In some embodiments, Myc is Myc-ER. In someembodiments, Myc is Myc-GR. In some embodiments, the organism is axenomouse.

In some embodiments, the organism (e.g., a mammal) is obtained by: (a)presenting an immuno-deficient organism (e.g., a mammal); and (b)administering to the organism (e.g., a mammal) a plurality ofhematopoietic stem cells that over-express MYC. In some embodiments, MYCencodes a fusion peptide. In some embodiments, MYC encodes a fusionpeptide comprising a PTD. In some embodiments, MYC encodes TAT-MYC. Insome embodiments, MYC encodes Myc-ER. In some embodiments, MYC encodesMyc-GR. In some embodiments, over-expression of MYC is induced bycontacting the cell with a small molecule, a biologic, a peptide, anantibody, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the cell (e.g., a mammalian cell) isderived from the plurality of hematopoietic stem cells that over-expressMYC. In some embodiments, over-expression of MYC is induced bycontacting the cell with a small molecule, a biologic, a peptide, anantibody, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the immuno-deficient organism (e.g., amammal) is obtained by irradiating the organism (e.g., a mammal). Insome embodiments, the immuno-deficient organism (e.g., a mammal) is aRag-1ko, Rag-2, SCID, DNA-PK, Ku70, Ku80, XRCC4, or μMT mouse. In someembodiments, the organism (e.g., a mammal) expresses the selectedantigen. In some embodiments, a method described herein furthercomprises recovering the antibody produced by the antibody producingcell. In some embodiments, a method described herein further comprisessubjecting the antibody producing cell to conditions that induceover-expression of MYC. In some embodiments, over-expression of MYC isinduced by contacting the cell with a small molecule, a biologic, apeptide, an antibody, or a combination thereof. In some embodiments, thesmall molecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, over-expression of MYC is induced bycontacting the cell with a shRNA molecule for Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the nucleic acid sequence(e.g., a transgenic sequence) that encodes the Myc peptide comprises aninducible promoter or a B-cell-selective promoter. In some embodiments,prior to isolating the antibody producing cell from the organism (e.g.,a mammal) the antibody producing cell is subjected to conditions thatinduce over-expression of MYC. In some embodiments, over-expression ofMYC is induced by contacting the cell with a small molecule, a biologic,a peptide, an antibody, or a combination thereof. In some embodiments,the small molecule is an antagonist of Max-1, Mxi-1, MAD, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1. In some embodiments, the small molecule is anantagonist of Mxi-1. In some embodiments, the small molecule is anantagonist of MAD. In some embodiments, over-expression of MYC isinduced by contacting the cell with a siRNA molecule for Max-1, Mxi-1,MAD, or a combination thereof. In some embodiments, over-expression ofMYC is induced by contacting the cell with a shRNA molecule for Max-1,Mxi-1, MAD, or a combination thereof.

In some embodiments, provided herein is a method of producing a human orhumanized antibody comprising:

-   -   a. providing an antibody producing human cell;    -   b. isolating a human gene that encodes the antibody from the        antibody producing human cell; and    -   c. introducing (or otherwise providing to) the human gene that        encodes the antibody into an cell (e.g., a mammalian cell) that        comprises nucleic acid sequence (e.g., a transgenic sequence)        that encodes an onco-peptide (e.g., recombinant onco-peptide;        e.g., Myc).

In some embodiments, the human gene encodes human IgH and IgL, whereinthe IgH and IgL together form an antibody that specifically binds theselected antigen. In some embodiments, the cell (e.g., a mammalian cell)is a B-cell. In some embodiments, a method described herein furthercomprises transplanting the cell (e.g., a mammalian cell) into a mouse.In some embodiments, the human gene isolated encodes a first antibodyand a second antibody. In some embodiments, a method described hereinfurther comprises recovering the antibody from the cell (e.g., amammalian cell). In some embodiments, the nucleic acid sequence (e.g., atransgenic sequence) that encodes the Myc peptide comprises an induciblepromoter or a B-cell-selective promoter. In some embodiments, the Mycpeptide is a Myc-ER fusion peptide. In some embodiments, the Myc peptideis a Myc-GR fusion peptide. In some embodiments, the Myc peptide is aTAT-Myc fusion peptide disclosed herein.

In some embodiments, provided herein is a method of producing a human orhumanized antibody comprising: (a) introducing (or otherwise providingto) at least one gene encoding a human immunoglobulin into a cell thatover-expresses Myc; and (b) isolating the encoded human immunoglobulin.

In some embodiments, over-expression of MYC is induced by contacting thecell with a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.In some embodiments, over-expression of MYC is induced by contacting thecell with a shRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof.

In some embodiments, peripheral blood B-cells are obtained from a human.In some embodiments, the human has a serum antibody titer to a certainantigen. The B-cells are obtained using standard approaches. In someembodiments, the B-cells are enriched or purified by any suitablemethod. In some embodiments, purified B-cells are panned on plasticplates coated with the selected antigen in order to enrich for thoseB-cells with a specificity of interest. In some embodiments, theselected antigen is conjugated to magnetic beads that are used toisolate the B-cells with the specificity of interest. In someembodiments, nucleic acids, that encode for the heavy and light chain ofan antibody, are isolated from enriched populations of antigen specificB-cells. In some embodiments, nucleic acids are isolated from purifiedantigen specific B-cells. In some embodiments, nucleic acids encodingthe heavy and light chain of an antibody are isolated by RT-PCR or PCR.In some embodiments, enriched populations of antigen specific B cellsare single-cell sorted into terasaki plates for single cell RT-PCR. Insome embodiments, nucleic acids encoding the variable regions for theheavy and light chain of an antibody are isolated.

In some embodiments, nucleic acids encoding human antibodies aretransferred into vectors to enable the expression of the humanantibodies in cells that over-express MYC. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments, thevectors are plasmids or viral vectors. In some embodiments, nucleicacids encoding the variable regions of a human antibody are transferredinto a vector cassette comprising the constant region of a humanantibody. Using this process or any other suitable process, a variety ofhuman antibodies with defined antigen specificity are produced of thedesired class, subclass or isotype. In some embodiments, cDNA fragmentsencoding human variable regions are cloned into retroviral vectors thatencode a human heavy and light chain constant region. In someembodiments, the resulting vectors encoding human antibodies areintroduced, by any suitable method, into hematopoietic stem cells thatover-express MYC. In some embodiments, over-expression of MYC is inducedby contacting the cell with a small molecule, a biologic, a peptide, anantibody, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. Immortalized B-cells are then produced by methods describedherein. In some embodiments, hematopoietic stem cells are derived fromMMTV-tTA/TRE-MYC/Rag-1 knock out mice, Eμ-MYC/Rag-1 knock out mice, orbone marrow from a Rag-1 knock out mouse that was transduced with aretrovirus directing the over-expression of MYC. In some embodiments,cells expressing human antibodies and Myc are used to reconstituteimmuno-deficient organisms and are subject to the protocols describedherein to produce antibody or immortal antibody producing B-cells. Theresulting cell lines obtained from this approach encode humanimmunoglobulin sequences specific for the protein of interest. In someembodiments, this is an approach for the generation of cocktails ofinhibitory antibodies for viral infections, tumors, bacteria and fungi,etc.

As disclosed herein, a number of methods are provided to produceantigen-specific non-human (e.g., mouse) antibodies from hematopoieticstem cells or B-cells that over-express MYC. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments,over-expression of MYC is induced by contacting the cell with a shRNAmolecule for Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, provided herein, antigen specific non-human antibodies arehumanized and the humanized antibodies are expressed in hematopoieticstem cells or B-cells that over-express MYC. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments,nucleic acids encoding the selected antigen-specific mouse antibodiesare isolated from any source by any suitable method. In someembodiments, the nucleic acids encoding the selected antigen-specificmouse antibodies are isolated from a hybridoma that expresses anantibody of interest. In some embodiments, the nucleic acids encodingthe selected antigen-specific non-human (e.g., mouse) antibodies areisolated from any antibody producing cell described herein. In someembodiments, the nucleic acids encoding the selected antigen-specificmouse antibodies are isolated from hematopoietic stem cells thatover-express MYC. In some embodiments, over-expression of MYC is inducedby contacting the cell with a small molecule, a biologic, a peptide, anantibody, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the nucleic acids encoding the selectedantigen-specific non-human (e.g., mouse) antibodies are isolated fromimmortalized B-cells that over-express MYC. In some embodiments,over-expression of MYC is induced by contacting the cell with a smallmolecule, a biologic, a peptide, an antibody, or a combination thereof.In some embodiments, the small molecule is an antagonist of Max-1,Mxi-1, MAD, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1. In some embodiments, the smallmolecule is an antagonist of Mxi-1. In some embodiments, the smallmolecule is an antagonist of MAD. In some embodiments, over-expressionof MYC is induced by contacting the cell with a siRNA molecule forMax-1, Mxi-1, MAD, or a combination thereof. In some embodiments,isolated nucleic acids that encode antigen-specific antibodies aregenetically altered to encode humanized, CDR engrafted, or chimericantibodies. In some embodiments, PCR is used to amplify the rearrangedVDJ joint sequence derived from the murine Ig heavy chain and VJ regionsfrom the Ig light chain loci. In some embodiments, the PCR-amplifiedfragments are cloned into a retroviral plasmid that encodes a human Igheavy chain and human Ig light chain. In some embodiments, thePCR-amplified fragments are cloned into any vector that directs theexpression of the desired antibody. In some embodiments, the Ig heavychain and light chain sequences are spaced by an IRES element, such thatboth cDNAs are expressed from the same viral vector. A number ofdifferent viral vectors are known in the art that enable the generationof different antibody isotypes, classes, and subclasses. In someembodiments, the sequences encoding the Fc regions are further modifiedto alter effector function, the ability to trigger autoimmune reactionsor the ability to induce immune-complex deposition problems.

In some embodiments, the resulting vectors or retroviruses areintroduced into hematopoietic stem cells that over-express MYC. In someembodiments, over-expression of MYC is induced by contacting the cellwith a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.In some embodiments, hematopoietic stem cells that over-express MYC areobtained from an organism that inducibly expresses recombinant Myc(e.g., MMTV-tTA/TRE-MYC/Rag-1 knock out mice). In some embodiments, thehematopoietic stem cells described herein are transferred into alethally irradiated animal, e.g., lethally irradiated C57/BL6 wild typemice. In some embodiments, the resulting mice generate immortalmonoclonal antibody producing cells that are antigen specific with allof the added features of the humanized antibodies.

In some embodiments, nucleic acids encoding human antibodies areisolated from human B-cells obtained from either healthy donors,patients who suffer from antibody-mediated autoimmune diseases (e.g.Sjögren's syndrome, Hashimoto's thyroiditis, Systemic LupusErythematosus, Waldenström's macroglobulinemia, etc), or patients whosuffer from Non-Hodgkin's lymphomas (e.g. Burkitt's lymphomas,Follicular Like Lymphomas, Diffuse Large B-cell lymphomas, MGUS andMultiple Myeloma). In certain embodiments, the nucleic acids encodinghuman antibodies are cloned, using any suitable manner, into retroviralvectors as described herein to produce retroviral libraries. In someembodiments, two different retroviral vectors are used to expedite thegeneration of the retroviral libraries. In some embodiments, theresulting vectors encoding human antibodies are introduced, using anysuitable manner, into hematopoietic stem cells that over-express MYC. Insome embodiments, over-expression of MYC is induced by contacting thecell with a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.In certain embodiments, such hematopoietic stem cells are thendifferentiated to produce immortalized B-cells (including conditionallyimmortalized) that express the selected antigen encoded by the nucleicacids cloned. In some embodiments, hematopoietic stem cells are derivedfrom an organism that conditionally expresses Myc (e.g.,MMTV-tTA/TRE-MYC/Rag-1 knock out mice), an organism that expressesrecombinant Myc in a B-cell (e.g., Eμ-MYC/Rag-1 knock out mice), or bonemarrow from an immuno-deficient animal (e.g., a Rag-1 knock out mouse)that was transduced with a retrovirus directing the over-expression ofMYC. In some embodiments, cells expressing human antibodies and Myc areused to reconstitute immuno-deficient organisms and are subject to theprotocols described herein to produce antibody or immortal antibodyproducing B-cells. In some embodiments, the retroviral libraries areused to transduce hematopoietic stem cells obtained from an organismthat conditionally expresses Myc (e.g., MMTV-tTA/TRE-MYC/Rag-1 knock outmice) in order to generate bone marrow chimeric mice. In someembodiments, the bone marrow chimeric mice only make B-cells thatexpress human antibodies and are maintained on a doxycycline containingdiet until they are ready for immunization (in order to suppress MYCexpression). In some embodiments, these mice are immunized in theabsence of doxycycline to induce over-expression of MYC in theirB-cells. In some embodiments, the reactive, antigen-specific B-cells areisolated and enriched by panning against an antigen. In someembodiments, reactive, antigen-specific B-cells are isolated andenriched by other selection methods known in the art. Examples ofselections methods include but are not limited to panning, cell sortingand magnetic bead isolation techniques. In some embodiments,antigen-specific populations are grown in the presence of Myc (e.g.,recombinant Myc) activity in order to produce immortal monoclonal celllines that generate human antibodies. In some embodiments,antigen-specific populations are grown in the presence of Mycover-expression in order to produce immortal monoclonal cell lines thatgenerate human antibodies. In some embodiments, the approach describedabove is used to specifically isolate human IgA antibodies to specificantigens. Human IgA antibodies are highly sought after for prophylaxis.In some embodiments, the Fc region of any antibody produced herein iseasily exchanged or altered to produce different classes, subclasses, orisotypes or antibodies with modified effector functions.

Onco-Peptides

In certain embodiments, any onco-peptide is suitable for use herein. Insome embodiments, the onco-peptide promotes cell viability, cellimmortality, cell growth and/or cell proliferation. In specificembodiments, an onco-peptide is, by way of non-limiting example, a Mycpeptide (e.g., a recombinant Myc peptide (e.g., a Myc fusion peptide asdescribed herein)), a Notch-1 peptide (e.g., a recombinant peptide), anAkt peptide (e.g., a recombinant peptide), an hTERT peptide (e.g., arecombinant peptide), and the like. Further examples of onco-peptides(e.g., peptides encoded by a protooncogene or an oncogene) are set forthin U.S. 2007/0116691, which is hereby incorporated by reference for suchdisclosures. In some embodiments, the Myc peptide is a fusion peptide.In some embodiments, the Myc peptide comprises a PTD. In someembodiments, the Myc peptide is a TAT-Myc peptide as described herein.In some embodiments, the Myc peptide is Myc-ER. In some embodiments, theMyc peptide is Myc-GR.

While in certain embodiments, described herein, Myc is the onco-peptideutilized, it is to be understood that any onco-peptide that promotescell viability, cell immortality, cell growth and/or cell proliferationis optionally substituted for the Myc peptide. In some embodiments,over-expression of an onco-peptide results in onco-peptide activity.

For example, Myc-over-expressing BCR^(HEL) transgenic B-cells mount avigorous response to soluble HEL (hen egg-white lysozyme) and engender apolyclonal autoimmune lymphoprolifeative disease prior to the onset of amalignancy (FIGS. 1-6) The over-expression of MYC in auto reactiveB-cells renders the B-cells independent of T-cell help, through Myc'sabilities to provide proliferative and survival signals. In someembodiments, the expanded population of Myc over-expressing, autoreactive B-cells develop into a B-cell lymphoma that remains dependentupon both continuous exposure to its cognate antigen and Myc (e.g.,recombinant Myc) activity. In some embodiments, B-cells are harvestedfrom, e.g., the lymph nodes, spleens and/or bone marrow of tumor-bearingorganisms. In certain embodiments, such B-cells are used to establishmany cell lines that expressed the BCR^(HEL) nucleic acid sequence(e.g., a transgenic sequence) and secrete anti-HEL antibody, withoutrequiring cell fusion of the primary cells to a fusion partner.

In some embodiments, the onco-peptide is a fusion peptide. In certainembodiments, fusion peptides include, e.g., a transduction domain and/ora receptor (e.g., Tat-Myc, Myc-ER, Myc-GR, Tat-Myc-ER, and Tat-Myc-GR).

In some embodiments, the onco-peptide is Bcl-2, Mcl-1, a Bcl-2 familymember or a derivative thereof. In some embodiments, the onco-peptide isRas, H-Ras, K-Ras, N-Ras, ERK, c-erbB2, RET or TRK or a derivativethereof. In some embodiments, provided herein is a method of producingan antibody specific for an antigen, comprising contacting a cell (e.g.,a mammalian cell) comprising a nucleic acid sequence (e.g., a transgenicsequence) that encodes SV40 T-Ag with a selected antigen. In certainembodiments, an onco-peptide of a method disclosed herein is optionallysubstituted with a growth factor that promotes cell viability, cellimmortality, cell growth and/or cell proliferation. In some embodiments,provided herein is a method of producing an antibody specific for anantigen, comprising contacting a cell (e.g., a mammalian cell)comprising a nucleic acid sequence (e.g., a transgenic sequence) thatencodes a growth factor receptor polypeptide with a selected antigen.Examples of growth factors receptors include but are not limited toepidermal growth factor receptor (EGFr), platelet derived growth factorreceptor (PDGFr), erbB2, erbB4, vascular endothelial growth factorreceptor (VEGFr), tyrosine kinase with immunoglobulin-like and epidermalgrowth factor homology domains (TIE-2), insulin growth factor-I (IGFI)receptor, macrophage colony stimulating factor (cfms), BTK, ckit, cmet,fibroblast growth factor (FGF) receptors, Trk receptors (TrkA, TrkB, andTrkC), ephrin (eph) receptors, and the RET protooncogene. In someembodiments, provided herein is a method of producing an antibodyspecific for an antigen, comprising contacting a cell (e.g., a mammaliancell) comprising a nucleic acid sequence (e.g., a transgenic sequence)that encodes a dominant negative tumor suppressor polypeptide with aselected antigen. In some embodiments, the dominant negative suppressorpolypeptide is a dominant negative of p53.

In some embodiments, provided herein is a method of producing anantibody specific for an antigen, comprising introducing (or otherwiseproviding to) a selected antigen into an organism (e.g., a mammal),wherein the organism (e.g., a mammal) comprises a nucleic acid sequence(e.g., a transgenic sequence) that encodes Bcl-2, Mcl-1, a Bcl-2 familymember, Ras, H-Ras, K-Ras, N-Ras, ERK, c-erbB2, RET or TRK, SV40 T-Ag,EGFr, PDGFr, erbB2, erbB4, VEGFr, TIE-2, IGFI receptor, cfms, BTK, ckit,cmet, an FGF receptor, a Trk receptor, an eph receptor, RET and/or adominant negative of p53. In some embodiments, the method furthercomprises generating immortal antigen-specific B-cells from the organism(e.g., a mammal). In some embodiments, the organism is a xenomouse. Insome embodiments, the method further comprises generating hematopoieticstem cells that are genetically altered to express at least one of thelisted nucleic acid sequences (e.g., a transgenic sequence). In someembodiments, a method disclosed herein further comprises the preparationof human antibodies. In some embodiments, the method further comprisesrecovering the antibody specific for an antigen from a plurality ofrecombinant B-cells generated by the expansion of the B-cell. In someembodiments, a method disclosed herein further comprises generatingantibody producing immortal, antigen-specific B-cells from an anergicB-cell that over-expresses at least one of the listed nucleic acidsequences (e.g., a transgenic sequence). In some embodiments, a methoddisclosed herein further comprises an antigen that is a self-antigen oran auto-antigen. In some embodiments, a method disclosed herein furthercomprises contacting a B-cell with the selected antigen andover-expressing the nucleic acid sequence (e.g., a transgenic sequence).In some embodiments, a method disclosed herein comprises immunizing theorganism (e.g., a mammal) with the selected antigen. In someembodiments, the organism (e.g., a mammal) further carries and expressesa nucleic acid sequence (e.g., a transgenic sequence) encoding theselected antigen. In some embodiments, a method disclosed herein furthercomprises a nucleic acid sequence (e.g., a transgenic sequence) encodinga Myc peptide (e.g., a recombinant Myc peptide (e.g., a Myc fusionpeptide as described herein)). In some embodiments, the Myc peptide is afusion peptide. In some embodiments, the Myc peptide comprises a PTD. Insome embodiments, the Myc peptide is a TAT-Myc peptide as describedherein. In some embodiments, the Myc peptide is Myc-ER. In someembodiments, the Myc peptide is Myc-GR.

In some embodiments, provided herein is a method of producing anantibody selective for an antigen, comprising (a) administering to animmunodeficient organism (e.g., a mammal) a plurality of hematopoieticstem cells that comprise a nucleic acid sequence (e.g., a transgenicsequence) encoding Bcl-2, Mcl-1, a Bcl-2 family member, Ras, H-Ras,K-Ras, N-Ras, ERK, c-erbB2, RET or TRK, SV40 T-Ag, EGFr, PDGFr, erbB2,erbB4, VEGFr, TIE-2, IGFI receptor, cfms, BTK, ckit, cmet, an FGFreceptor, a Trk receptor, an eph receptor, RET and/or a dominantnegative of p53; and (b) introducing (or otherwise providing to) aselected antigen into the immuno-deficient organism (e.g., a mammal). Insome embodiments, the organism is a xenomouse. In some embodiments, thehematopoietic stem cells further comprise a nucleic acid sequence (e.g.,a transgenic sequence) encoding a Myc peptide. In some embodiments, theMyc peptide is a fusion peptide. In some embodiments, the Myc peptidecomprises a PTD. In some embodiments, the Myc peptide is a TAT-Mycpeptide as described herein. In some embodiments, the Myc peptide isMyc-ER. In some embodiments, the Myc peptide is Myc-GR. In someembodiments, a method disclosed herein further comprises inducing aplurality of the hematopoietic stem cells to differentiate into B-cells.In some embodiments, a method described herein further comprisesrecovering a plurality of the B-cells that express the antibodyselective for the selected antigen from the immuno-deficient organism(e.g., a mammal). In some embodiments, a method described herein furthercomprises recovering the antibody from the plurality of B-cells thatexpress the antibody. In some embodiments, the nucleic acid sequence(e.g., a transgenic sequence) comprises an inducible promoter or aB-cell-selective promoter. In some embodiments, the nucleic acidsequence (e.g., a transgenic sequence) comprises an inducible promotercomprising one or more TREs. In some embodiments, the hematopoietic stemcells express tTA or rtTA. In some embodiments, a method describedherein providing doxycycline, tetracycline, or an analog thereof to theimmuno-deficient organism (e.g., a mammal) for a period of timesufficient to suppress the tTA-dependent transactivation, andwithdrawing the doxycycline, tetracycline, or analog thereof after theperiod to induce tTA-dependent transactivation. In some embodiments, thenucleic acid sequence (e.g., a transgenic sequence) comprises aB-cell-selective promoter and the B-cell selective promoter is the Eμpromoter. In some embodiments, the organism or cell further comprises anucleic acid sequence (e.g., a transgenic sequence) encoding a fusionpeptide. In some embodiments, the selected antigen is a self-antigen oran auto antigen. In some embodiments, the immuno-deficient organism(e.g., a mammal) is obtained by irradiating the organism (e.g., amammal). In some embodiments, the immuno-deficient organism (e.g., amammal) is a Rag-1ko, Rag-2, SCID, DNA-PK, Ku70, Ku80, XRCC4, or μMTmouse. In some embodiments, the immuno-deficient organism (e.g., amammal) expresses the selected antigen. In some embodiments, theimmuno-deficient organism (e.g., a mammal) that expresses the selectedantigen is transgenic for the selected antigen. In some embodiments, theselected antigen is introduced by transfection with a nucleic acidexpression vector or infection with a recombinant virus expressionvector. In some embodiments, the recombinant virus expression vector isa recombinant lentivirus.

In some embodiments, provided herein is a method of producing a human orhumanized antibody comprising: (a) providing a cell (e.g., a mammaliancell) that expresses human antibodies and comprises nucleic acidsequence (e.g., a transgenic sequence) that encodes a recombinant Bcl-2,Mcl-1, a Bcl-2 family member, Ras, H-Ras, K-Ras, N-Ras, ERK, c-erbB2,RET or TRK, SV40 T-Ag, EGFr, PDGFr, erbB2, erbB4, VEGFr, TIE-2, IGFIreceptor, cfms, BTK, ckit, cmet, an FGF receptor, a Trk receptor, an ephreceptor, RET and/or a dominant negative p53 polypeptide; and (b)contacting the cell (e.g., a mammalian cell) with a selected antigen. Insome embodiments, the cell (e.g., a mammalian cell) that expresses humanantibodies and comprises nucleic acid sequence (e.g., a transgenicsequence) that encodes a recombinant Bcl-2, Mcl-1, a Bcl-2 familymember, Ras, H-Ras, K-Ras, N-Ras, ERK, c-erbB2, RET or TRK, SV40 T-Ag,EGFr, PDGFr, erbB2, erbB4, VEGFr, TIE-2, IGFI receptor, cfms, BTK, ckit,cmet, an FGF receptor, a Trk receptor, an eph receptor, RET and/or adominant negative p53 polypeptide is a B-cell. In some embodiments, thecell (e.g., a B-cell) inducibly over-expresses Myc. In some embodiments,the cell (e.g., a B-cell) inducibly over-expresses the recombinantBcl-2, Mcl-1, a Bcl-2 family member, Ras, H-Ras, K-Ras, N-Ras, ERK,c-erbB2, RET or TRK, SV40 T-Ag, EGFr, PDGFr, erbB2, erbB4, VEGFr, TIE-2,IGFI receptor, cfms, BTK, ckit, cmet, an FGF receptor, a Trk receptor,an eph receptor, RET and/or dominant negative p53 polypeptide. In someembodiments, the cell (e.g., a mammalian cell) that expresses humanantibodies and comprises nucleic acid sequence (e.g., a transgenicsequence) a that encodes Bcl 2, Mcl-1, a Bcl-2 family member, Ras,H-Ras, K-Ras, N-Ras, ERK, c-erbB2, RET or TRK, SV40 T-Ag, EGFr, PDGFr,erbB2, erbB4, VEGFr, TIE-2, IGFI receptor, cfms, BTK, ckit, cmet, an FGFreceptor, a Trk receptor, an eph receptor, RET and/or dominant negativep53 polypeptide is present in an organism (e.g., a mammal), and whereinthe method further comprises recovering the antibody producing cell fromthe organism (e.g., a mammal). In some embodiments, the organism is axenomouse. In some embodiments, the organism (e.g., a mammal) is amouse. In some embodiments, the organism (e.g., a mammal) utilized in amethod described herein is obtained by: (a) presenting animmuno-deficient organism (e.g., a mammal); and (b) administering to theorganism (e.g., a mammal) a plurality of hematopoietic stem cells thatover-express Bcl-2, Mcl-1, a Bcl-2 family member, Ras, H-Ras, K-Ras,N-Ras, ERK, c-erbB2, RET or TRK, SV40 T-Ag, EGFr, PDGFr, erbB2, erbB4,VEGFr, TIE-2, IGFI receptor, cfms, BTK, ckit, cmet, an FGF receptor, aTrk receptor, an eph receptor, RET and/or a dominant negative p53polypeptide. In some embodiments, the immuno-deficient organism (e.g., amammal) is obtained by irradiating the organism (e.g., a mammal). Insome embodiments, the immuno-deficient organism (e.g., a mammal) is aRag-1ko, Rag-2, SCID, DNA-PK, Ku70, Ku80, XRCC4, or μMT mouse. In someembodiments, the organism (e.g., a mammal) expresses the selectedantigen. In some embodiments, a method described herein furthercomprises recovering the antibody produced by the antibody producingcell or animal. In some embodiments, a method described herein furthercomprises subjecting the antibody producing cell to conditions thatinduce over-expression of Bcl-2, Mcl-1, a Bcl-2 family member, Ras,H-Ras, K-Ras, N-Ras, ERK, c-erbB2, RET or TRK, SV40 T-Ag, EGFr, PDGFr,erbB2, erbB4, VEGFr, TIE-2, IGFI receptor, cfms, BTK, ckit, cmet, an FGFreceptor, a Trk receptor, an eph receptor, RET and/or a dominantnegative p53 polypeptide.

In some embodiments, provided herein is a method of producing a human orhumanized antibody comprising: (a) providing an antibody producing humancell; (b) isolating a human gene that encodes the antibody from theantibody producing human cell; and (c) introducing (or otherwiseproviding to) the human gene that encodes the antibody into an cell(e.g., a mammalian cell) that comprises nucleic acid sequence (e.g., atransgenic sequence) that encodes a Bcl-2, Mcl-1, a Bcl-2 family member,Ras, H-Ras, K-Ras, N-Ras, ERK, c-erbB2, RET or TRK, SV40 T-Ag, EGFr,PDGFr, erbB2, erbB4, VEGFr, TIE-2, IGFI receptor, cfms, BTK, ckit, cmet,an FGF receptor, a Trk receptor, an eph receptor, RET and/or a dominantnegative p53 polypeptide. In some embodiments, the cell (e.g., amammalian cell) over-expresses a Myc peptide (e.g., a recombinant Mycpeptide (e.g., a Myc fusion peptide as described herein)). In someembodiments, the Myc peptide is a fusion peptide. In some embodiments,the Myc peptide is a TAT-Myc peptide as described herein. In someembodiments, over-expression of MYC is induced by contacting the cellwith a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.In some embodiments, over-expression of MYC is induced by contacting thecell with a shRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the human gene encodes human IgH and IgL,wherein the IgH and IgL together form an antibody that specificallybinds the selected antigen. In some embodiments, the cell (e.g., amammalian cell) is a B-cell. In some embodiments, a method describedherein further comprises transplanting the cell (e.g., a mammalian cell)into a mouse. In some embodiments, the human gene isolated encodes afirst antibody and a second antibody. In some embodiments, a methoddescribed herein further comprises recovering the antibody from the cell(e.g., a mammalian cell).

In some embodiments, provided herein is a method of producing a human orhumanized antibody comprising: (a) introducing (or otherwise providingto) at least one gene encoding a human immunoglobulin into a cell thatover-expresses a Bcl-2, Mcl-1, a Bcl-2 family member, Ras, H-Ras, K-Ras,N-Ras, ERK, c-erbB2, RET or TRK, SV40 T-Ag, EGFr, PDGFr, erbB2, erbB4,VEGFr, TIE-2, IGFI receptor, cfms, BTK, ckit, cmet, an FGF receptor, aTrk receptor, an eph receptor, RET and/or a dominant negative p53polypeptide; and (b) isolating the encoded human immunoglobulin.

In some embodiments, provided herein is an isolated B-cell for producinga human or humanized antibody, wherein the B-cell over-expresses aBcl-2, Mcl-1, a Bcl-2 family member, Ras, H-Ras, K-Ras, N-Ras, ERK,c-erbB2, RET or TRK, SV40 T-Ag, EGFr, PDGFr, erbB2, erbB4, VEGFr, TIE-2,IGFI receptor, cfms, BTK, ckit, cmet, an FGF receptor, a Trk receptor,an eph receptor, RET and/or a dominant negative p53 polypeptide. In someembodiments, the B-cell also over-expresses a Myc peptide (e.g., arecombinant Myc peptide (e.g., a Myc fusion peptide as describedherein)). In some embodiments, the Myc peptide is a fusion peptide. Insome embodiments, the Myc peptide is a TAT-Myc peptide as describedherein. In some embodiments, over-expression of MYC is induced bycontacting the cell with a small molecule, a biologic, a peptide, anantibody, or a combination thereof. In some embodiments, the smallmolecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, over-expression of MYC is induced bycontacting the cell with a shRNA molecule for Max-1, Mxi-1, MAD, or acombination thereof.

Tolerance

Onco-peptide (e.g., recombinant onco-peptide; e.g., Myc) activity, insome embodiments, terminates (or reduces) B-cell tolerance to an antigen(e.g., a soluble antigen). Therefore, in some embodiments, of a methoddescribed herein, the cell (e.g., a mammalian cell) comprising a nucleicacid sequence (e.g., a transgenic sequence) encoding an onco-peptide istolerant and/or anergic to the selected antigen.

In some embodiments, provided herein is a method of producing anantibody and/or cell that produces an antibody that specifically bindsto an antigen, comprising: contacting a cell comprising a nucleic acidsequence (e.g., a transgenic sequence) that encodes an onco-peptide(e.g., recombinant onco-peptide; e.g., Myc) with a selected antigen,wherein in the absence of onco-peptide activity (expression and/oronco-peptide function), the cell (e.g., a mammalian cell) is tolerantand/or anergic to the selected antigen.

In some embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide comprises a PTD. In some embodiments, theonco-peptide is a TAT-Myc peptide as described herein. In someembodiments, the onco-peptide is Myc-ER. In some embodiments, theonco-peptide is Myc-GR.

In certain embodiments, the method further comprises recovering one ormore B-cells that express the antibody that specifically binds to theselected antigen. In specific embodiments, the selected antigen is aself antigen.

In some embodiments, provided herein is a method of producing anantibody that specifically binds to an antigen comprising:

-   -   a. inducing onco-peptide (e.g., recombinant onco-peptide; e.g.,        Myc) activity (expression and/or onco-peptide function) in a        cell (e.g., B-cell) that expresses the selected antibody and        comprises an onco-peptide that is inducibly activated (e.g.,        Myc-ER, Myc-GR) and/or a nucleic acid sequence (e.g., a        transgenic sequence) that encodes an onco-peptide (e.g.,        TRE-Myyc, TRE-Myc-ER, or TRE-Myc-GR);    -   b. recovering the antibody expressed from the organism cell        (e.g., B-cell).

In some embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide comprises a PTD. In some embodiments, theonco-peptide is a TAT-Myc peptide as described herein. In someembodiments, the onco-peptide is Myc-ER. In some embodiments, theonco-peptide is Myc-GR.

In some embodiments, the organism is a xenomouse.

In some embodiments, in the absence of onco-peptide (activity(expression and/or onco-peptide function), the cell (e.g., a mammaliancell) is tolerant and/or anergic to the selected antigen. In someembodiments, the Myc peptide is a fusion peptide. In some embodiments,the Myc peptide is a TAT-Myc peptide as described herein. In certainembodiments, induction of onco-peptide activity induces proliferation ofthe cell (e.g., a mammalian cell). Thus, in certain embodiments, theantibody is recovered from a plurality of cells (e.g., B-cells)generated by the expansion of the cell. In specific embodiments, theselected antigen is a self antigen. In some embodiments, the process ofrecovering an antibody comprises recovering the nucleic acids thatencode said antibody.

In some embodiments, provided herein is a method of producing anantibody that specifically binds to a self-antigen comprising:

-   -   a. inducing onco-peptide (e.g., recombinant onco-peptide; e.g.,        Myc) activity (expression and/or onco-peptide function) in a        cell (e.g., B-cell) that expresses the selected antibody and        comprises a onco-peptide that is inducibly activated (e.g.,        Myc-ER, or Myc-GR) and/or a nucleic acid sequence (e.g., a        transgenic sequence) that encodes an onco-peptide (e.g.,        TRE-Myc, TRE-Myc-GR, or TRE-Myc-ER);    -   b. recovering the antibody expressed from cell (e.g., B-cell).

In some embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide comprises a PTD. In some embodiments, theonco-peptide is a TAT-Myc peptide as described herein. In someembodiments, the onco-peptide is Myc-ER. In some embodiments, theonco-peptide is Myc-GR.

In some embodiments, provided herein is a method of producing anantibody that specifically binds to a self-antigen comprising:

-   -   a. contacting a cell that expresses the antibody with an        onco-peptide (e.g., recombinant onco-peptide; e.g., Myc,        TAT-Myc);    -   b. recovering the antibody expressed from cell (e.g., B-cell).

In some embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide comprises a PTD. In some embodiments, theonco-peptide is a TAT-Myc peptide as described herein. In someembodiments, the onco-peptide is Myc-ER. In some embodiments, theonco-peptide is Myc-GR.

In some embodiments, the method comprises: contacting a cell thatexpresses the antibody with a TAT-Myc fusion peptide disclosed herein;and recovering the antibody expressed from cell (e.g., B-cell).

In some embodiments, in the absence of onco-peptide activity (expressionand/or onco-peptide function), the cell (e.g., a mammalian cell) istolerant and/or anergic to the self antigen.

In some embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide comprises a PTD. In some embodiments, theonco-peptide is a TAT-Myc peptide as described herein. In someembodiments, the onco-peptide is Myc-ER. In some embodiments, theonco-peptide is Myc-GR.

In certain embodiments, induction of onco-peptide activity inducesproliferation of the cell (e.g., a mammalian cell). Thus, in certainembodiments, the antibody is recovered from a plurality of cells (e.g.,B-cells) generated by the expansion of the cell.

In some embodiments, provided herein is a method of producing anantibody selective for an antigen, comprising, (a) inducing Myc (e.g.,recombinant Myc) activity in a tolerant B-cell that comprises a Mycpeptide or a nucleic acid sequence (e.g., a transgenic sequence)encoding a Myc peptide; (b) expanding the B-cell after the induction;and (c) recovering the expressed antibody that is selective for theselected antigen from a plurality of recombinant B-cells generated bythe expansion of the B-cell.

In some embodiments, the Myc peptide is a fusion peptide. In someembodiments, the Myc peptide comprises a PTD. In some embodiments, theMyc peptide is a TAT-Myc peptide as described herein. In someembodiments, the Myc peptide is Myc-ER. In some embodiments, the Mycpeptide is Myc-GR.

In some embodiments, provided herein is a method of producing anantibody selective for an antigen, comprising, (a) inducing Myc (e.g.,recombinant Myc) activity in a B-cell that expresses the antibody, andcomprises a Myc peptide or a nucleic acid sequence (e.g., a transgenicsequence) encoding the Myc peptide; (b) expanding the B-cell after theinduction; and (c) recovering the expressed antibody from a plurality ofrecombinant B-cells generated by the expansion of the B-cell.

In some embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide comprises a PTD. In some embodiments, theonco-peptide is a TAT-Myc peptide as described herein. In someembodiments, the onco-peptide is Myc-ER. In some embodiments, theonco-peptide is Myc-GR.

In certain embodiments, expansion of a cell (e.g., a mammalian cell or aB-cell) described in the methods herein is caused by the induction ofthe onco-peptide (e.g., recombinant onco-peptide; e.g., Myc) activity inthe cell (e.g., B-cell).

For example, Myc over-expressing Ars.A1 mice break tolerance and developactivated antigen-specific B-cells. Ars.A1 mice express a transgenicB-cell receptor (BCR) specific for the selected antigen arsenate (Ars).In these mice the transgenic BCR specifically binds to arsenate, butexhibits low affinity cross-reactivity with DNA Immature B cells fromthese organisms are anergic as a consequence of Ag recognition in thebone marrow. Eμ-MYC mice express Myc almost exclusively in B-cells underthe control of the immunoglobulin heavy chain gene enhancer. Micederived from a cross between the Ars.A1 mouse and the Eμ-MYC straindevelop a Burkitt's like lymphoma. The tumors are composed of mature,activated B-cells that express arsenate-specific IgM on their surface.Thus, as disclosed herein, in some embodiments, Myc (e.g., recombinantMyc) activity breaks tolerance for auto reactive B-cells in the contextof a low-affinity, anti-DNA antibody. In some embodiments, Myc (e.g.,recombinant Myc) activity breaks tolerance of B-cells that bind selfantigen or auto antigens. In some embodiments, provided herein is amethod of producing an antibody that specifically binds to an antigen,comprising, (a) inducing Myc (e.g., recombinant Myc) activity in aB-cell that binds the selected antigen, and comprises a nucleic acidsequence (e.g., a transgenic sequence) encoding the recombinant Mycpeptide; (b) expanding the B-cell after the induction; and (c)recovering the expressed antibody from a plurality of recombinantB-cells generated by the expansion of the B-cell.

In some embodiments, the onco-peptide is a fusion peptide. In someembodiments, the onco-peptide comprises a PTD. In some embodiments, theonco-peptide is a TAT-Myc peptide as described herein. In someembodiments, the onco-peptide is Myc-ER. In some embodiments, theonco-peptide is Myc-GR.

In some embodiments, the nucleic acid sequence (e.g., a transgenicnucleic acid sequence) encoding the Myc peptide comprises a B-cellselective promoter.

In some embodiments, provided herein is a method of producing anantibody specific for an antigen, comprising contacting a cellcomprising a nucleic acid sequence (e.g., a transgenic sequence) thatencodes a Myc peptide with a selected antigen. In some embodiments, theMyc peptide is a fusion peptide. In some embodiments, the Myc peptidecomprises a PTD. In some embodiments, the Myc peptide is a TAT-Mycpeptide as described herein. In some embodiments, the Myc peptide isMyc-ER. In some embodiments, the Myc peptide is Myc-GR.

In some embodiments, the cell (e.g., a mammalian cell) is a B-cell or acell that expresses a B cell receptor on its cell surface. In someembodiments, the cell (e.g., a mammalian cell) expresses CD79 on itscells surface. In some embodiments, the cell (e.g., a mammalian cell) isa mammalian cell. In some embodiments, the cell (e.g., a mammalian cell)is a B-cell with an intact salvage pathway for purine biosynthesis.

In some embodiments, the nucleic acid sequence (e.g., a transgenicsequence) that encodes a Myc peptide comprises a B-cell-selectivepromoter operably linked to an open reading frame of the encoded Mycpeptide. In some embodiments, the B-cell selective promoter is the Eμpromoter. In some embodiments, the organism (e.g., a mammal) furthercomprises a nucleic acid sequence (e.g., a transgenic sequence) encodingthe selected antigen.

In some embodiments, provided herein is a method of producing anantibody specific for an antigen, comprising (a) contacting a CD79expressing cell with a selected antigen wherein the CD79 expressing cellcomprises Myc, and (b) recovering a CD79 expressing cell that producesan antibody that specifically binds to the selected antigen.

In some embodiments, the Myc peptide is a fusion peptide. In someembodiments, the Myc peptide comprises a PTD. In some embodiments, theMyc peptide is a TAT-Myc peptide as described herein. In someembodiments, the Myc peptide is Myc-ER. In some embodiments, the Mycpeptide is Myc-GR.

In some embodiments, the recovered CD79 expressing cell that produces anantibody that specifically binds to the selected antigen maintains thesame number of chromosomes throughout the process. In some embodiments,the CD79 expressing cell that produces an antibody that specificallybinds to the selected antigen produces soluble antibody. In someembodiments, the recovered CD79 expressing cell that produces anantibody that specifically binds to the selected antigen is expanded invitro and the antibody that specifically binds to the selected antigenis isolated. In some embodiments, the isolated antibody is purified.

In some embodiments, an organism provided in a method disclosed hereinis an organism with a B-cell specific expression of an onco-peptideand/or a temporally regulated over-expression of a polypeptide. In someembodiments, the organism is a xenomouse. In certain embodiments, a cellutilized in any process described herein is a cell from such anorganism. For example, MMTV-tTA/TRE-MYC mice enable B-cell specific,temporally regulated over-expression of MYC following the withdrawal ofdoxycycline from the diet. At four months of age and after withdraw ofdoxycycline, mice derived from a cross between the Ars.A1 mouse and theMMTV-tTA/TRE-MYC mouse generate activated peripheral B-cells andanti-nuclear antibodies. These mice also accumulate immune complexdeposits in their kidneys and develop B-cell lymphomas. Generation ofimmortal antibody producing cell lines from the tumors of these micedoes not require cell fusion with a myeloma partner cell.

In some embodiments, any Myc over-expressing organism or cell isutilized in a method described herein. In some embodiments, the organismis a xenomouse. In some embodiments, organisms that over-express MYCpredominantly in the B cell population, such as, for example, the Eμ-MYCmouse strain, are utilized.

In some embodiments, organisms or cells that over-express MYC in aninducible manner are utilized. In some embodiments, the organism is axenomouse. In some embodiments, the Myc over-expression is regulated ina temporal manner and in some embodiments, Myc over-expression issuppressed until the production of the antibodies commences. In someembodiments, the invention comprises the use of the MMTV-tTA/TRE-MYCmouse strain. In MMTV-tTA/TRE-MYC mice, Myc over-expression is commencedby removing the doxycycline or tetracycline from the diet of the mice ormouse cells. In some embodiments, the MMTV-tTA/TRE-MYC mouse strain isadministered doxycycline or tetracycline from birth until they are to beused to produce the antibody of interest, thereby minimizing theformation of spontaneous lymphoproliferative diseases.

In some embodiments, MMTV-rtTA/TRE-MYC mouse strains are utilized. InMMTV-rtTA/TRE-MYC mice, Myc over-expression is commenced by the additionof doxycycline or tetracycline to the diet of the mice.

In some embodiments, Myc-ER mice or mouse cells are utilized. In Myc-ERmice and mouse cells, Myc is activated by the addition of estrogen or ananalogue of estrogen. In some embodiments, the analogue of estrogen is4OHT. In some embodiments, alternative inducible geneexpression/repression systems are utilized. In some embodiments, anyanimal that over-expresses Myc is used to produce immortalized antibodyproducing cells by methods described herein.

In some embodiments, Myc-GR mice or mouse cells are utilized. In Myc-GRmice and mouse cells, Myc is activated by the addition of glucocorticoidor an analogue of glucocorticoid. In some embodiments, alternativeinducible gene expression/repression systems are utilized. In someembodiments, any animal that is genetically altered to over-express MYCis used to produce immortalized antibody producing cells by methodsdescribed herein.

In some embodiments, antigen responsive immortal B-cell lines aregenerated from anergic AN1/T3 cell populations found within an organismthat over-expresses MYC. In some embodiments, MYC encodes a fusionpeptide. In some embodiments, MYC encodes a fusion peptide comprising aPTD. In some embodiments, MYC encodes a TAT-Myc peptide as describedherein. In some embodiments, MYC encodes Myc-ER. In some embodiments,MYC encodes Myc-GR.

In some embodiments, over-expression of MYC is induced by contacting thecell with a small molecule, a biologic, a peptide, an antibody, or acombination thereof. In some embodiments, the small molecule is anantagonist of Max-1, Mxi-1, MAD, or a combination thereof. In someembodiments, the small molecule is an antagonist of Max-1. In someembodiments, the small molecule is an antagonist of Mxi-1. In someembodiments, the small molecule is an antagonist of MAD. In someembodiments, over-expression of MYC is induced by contacting the cellwith a siRNA molecule for Max-1, Mxi-1, MAD, or a combination thereof.

In some embodiments, these organisms express the selected antigen ofinterest. In some embodiments, the selected antigen is expressed byretroviral-mediated transduction of bone marrow derived hematopoieticstem cells (hematopoietic stem cells) with a recombinant retrovirusdirecting the expression of the selected antigen, using a standardtransgenic approach, or any other method for gene delivery intoorganisms. In some embodiments, the organisms are exposed to theselected antigen by immunization. In some embodiments, the organismsgenetically altered to express Myc are MMTV-tTA/TRE-MYC organisms thatare maintained on doxycycline from conception. In some embodiments, theMMTV-tTA/TRE-MYC organisms are MMTV-tTA/TRE-MYC mice.

In some embodiments, lymphocytes comprising the AN1/T3 cell populationsare isolated from the spleens of organisms genetically altered toexpress Myc and are further cultured in vitro. In some embodiments, thelymphocytes comprising the AN1/T3 populations are isolated from theblood, bone marrow, or lymph nodes. In some embodiments, lymphocytescomprising the AN1/T3 cell populations are cultured under conditions inwhich MYC is over-expressed. In some embodiments, lymphocytes comprisingthe AN1/T3 cell populations are cultured in the presence of antigenunder conditions in which MYC is over-expressed. In some embodiments,lymphocytes comprising the AN1/T3 cell populations are cultured in thepresence of antigen under conditions in which MYC is over-ecpressed. Insome embodiments, lymphocytes comprising the AN1/T3 cell populations arecultured in the presence of antigen and Myc (e.g., recombinant Myc)activity. In some embodiments, lymphocytes comprising the AN1/T3 cellpopulations are cultured in the presence of doxycycline. In someembodiments, the doxycycline concentration is from about 0.01 nM toabout 300 nM. In some embodiments, the doxycycline concentration is fromabout 1 nM to about 100 nM. In some embodiments, the doxycyclineconcentration is from 40 nM to 60 nM. In some embodiments, tetracycline,or an analogue of tetracycline, is used in place of doxycycline.

In some embodiments, antigen-specific B-cells are selected and isolatedby various suitable methods. A multitude of cell selection and cellisolation suitable techniques are available including, but are notlimited to flow cytometry, panning, magnetic beads, affinity columns,immunoprecipitation and various means of cell sorting.

In some embodiments, the cultured cells are re-exposed to antigen undercondition in which a Myc peptide (e.g., a recombinant Myc peptide) isactivated. In some embodiments, the Myc peptide is a fusion peptide. Insome embodiments, the Myc peptide comprises a PTD. In some embodiments,the Myc peptide is a TAT-Myc peptide as described herein. In someembodiments, the Myc peptide is a Myc-ER peptide as described herein. Insome embodiments, the Myc peptide is a Myc-GR peptide as describedherein.

In some embodiments, the selected antigen-specific B-cells or cells thatexpress an antigen-specific antibody are cloned and expanded without theneed for cell fusion to a partner cell.

In some embodiments, provided herein is a method of producing anantibody specific for an antigen, comprising (a) providing an AN1/T3B-cell wherein the AN1/T3 B-cell comprises a nucleic acid sequence(e.g., a transgenic sequence) encoding a Myc peptide, (b) contacting theAN1/T3 B-cell with an antigen wherein the AN1/T3 B-cell bindsspecifically to the selected antigen; and (c) inducing Myc (e.g.,recombinant Myc) activity in the AN1/T3 B-cell.

In some embodiments, the Myc peptide is a fusion peptide. In someembodiments, the Myc peptide comprises a PTD. In some embodiments, theMyc peptide is a TAT-Myc peptide as described herein. In someembodiments, the Myc peptide is a Myc-ER peptide as described herein. Insome embodiments, the Myc peptide is a Myc-GR peptide as describedherein.

In some embodiments, a method described herein further comprisesrecovering the antibody that specifically binds to the selected antigenfrom a plurality of the AN1/T3 B-cells. In some embodiments, the AN1/T3B-cell is present in an organism (e.g., a mammal) and inducing of Myc(e.g., recombinant Myc) activity in the AN1/T3 B-cell occurs in vivo. Insome embodiments, the contacting the AN1/T3 B-cell with an antigenoccurs in vitro or ex vivo.

In some embodiments, a method described herein further comprisesintroducing (or otherwise providing to) a Myc peptide or a nucleic acidsequence (e.g., a transgenic sequence) encoding the Myc peptide inot theAN1/T3 B-cell ex vivo prior to the inducing step. In some embodiments,the Myc peptide is a fusion peptide. In some embodiments, the Mycpeptide comprises a PTD. In some embodiments, the Myc peptide is aTAT-Myc peptide as described herein. In some embodiments, the Mycpeptide is a Myc-ER peptide as described herein. In some embodiments,the Myc peptide is a Myc-GR peptide as described herein.

In some embodiments, a method described herein comprises introducing (orotherwise providing to) the Myc peptide into the AN1/T3 B-cell ex vivo.In some embodiments, the Myc peptide is a fusion peptide. In someembodiments, the Myc peptide comprises a PTD. In some embodiments, theMyc peptide is a TAT-Myc peptide as described herein. In someembodiments, the Myc peptide is a Myc-ER peptide as described herein. Insome embodiments, the Myc peptide is a Myc-GR peptide as describedherein.

In some embodiments, the AN1/T3 B-cell is an anergic B-cell prior toinduction of Myc (e.g., recombinant Myc) activity. In some embodiments,the selected antigen is a self-antigen. In some embodiments, the nucleicacid sequence (e.g., a transgenic nucleic acid sequence) encoding theMyc peptide comprises a B-cell specific promoter operably linked to theopen reading frame encoding the Myc peptide. In some embodiments, thenucleic acid sequence (e.g., a transgenic nucleic acid sequence)encoding the Myc peptide comprises an inducible promoter operably linkedto the open reading frame for the Myc peptide. In some embodiments, theinducible promoter comprises one or more TREs and the recombinant AN1/T3B-cell further expresses a tTA peptide or an rtTA peptide. In someembodiments, the recombinant AN1/T3 B-cell expresses the tTA peptide.

In some embodiments, the Myc peptide is a Myc-ER fusion peptide. In someembodiments, inducing Myc (e.g., recombinant Myc) activity comprisescontacting the recombinant AN1/T3 B-cell with an ER ligand. In someembodiments, the Myc peptide is a Myc-GR fusion peptide. In someembodiments, inducing Myc (e.g., recombinant Myc) activity comprisescontacting the recombinant AN1/T3 B-cell with a GR ligand. In someembodiments, the AN1/T3 B-cell is a mouse cell. In some embodiments,contacting the AN1/T3 B-cell with an antigen comprises immunizing anorganism with the selected antigen.

In some embodiments, the immunized organism is an organism (e.g., amammal) carrying the nucleic acid sequence (e.g., a transgenic nucleicacid sequence) encoding the Myc peptide. In some embodiments, thenucleic acid sequence (e.g., a transgenic sequence) is an induciblenucleic acid sequence (e.g., a transgenic sequence). In someembodiments, the inducible nucleic acid sequence (e.g., a transgenicsequence) comprises a promoter, and wherein the promoter comprises oneor more TREs. In some embodiments, the organism (e.g., a mammal) furthercarries and expresses a tTA nucleic acid sequence (e.g., a transgenicsequence) or an rtTA nucleic acid sequence (e.g., a transgenicsequence). In some embodiments, the organism (e.g., a mammal) furthercarries and expresses a nucleic acid sequence (e.g., a transgenicsequence) encoding the selected antigen.

In some embodiments, provided herein is a method of producing anantibody specific for an antigen, comprising (a) providing an anergic ortolerant B-cell wherein the anergic or tolerant B-cell comprises anucleic acid sequence (e.g., a transgenic sequence) encoding a Mycpeptide, (b) contacting the anergic or tolerant B-cell with an antigenwherein the anergic or tolerant B-cell binds specifically to theselected antigen; and (c) inducing Myc (e.g., recombinant Myc) activityin the anergic or tolerant B-cell.

In some embodiments, the Myc peptide is a fusion peptide. In someembodiments, the Myc peptide comprises a PTD. In some embodiments, theMyc peptide is a TAT-Myc peptide as described herein. In someembodiments, the Myc peptide is a Myc-ER peptide as described herein. Insome embodiments, the Myc peptide is a Myc-GR peptide as describedherein.

In some embodiments, a method described herein further comprisesrecovering the antibody that specifically binds to the selected antigenfrom a plurality of the anergic or tolerant B-cells. In someembodiments, the anergic or tolerant B-cell is present in an organism(e.g., a mammal) and inducing of Myc (e.g., recombinant Myc) activity inthe anergic or tolerant B-cell occurs in vivo. In some embodiments, thecontacting the anergic or tolerant B-cell with an antigen occurs invitro or ex vivo.

In some embodiments, a method described herein further comprisesintroducing (or otherwise providing to) a Myc peptide or a nucleic acidsequence (e.g., a transgenic sequence) encoding the Myc peptide inot theanergic or tolerant B-cell ex vivo prior to the inducing step. In someembodiments, the Myc peptide is a fusion peptide. In some embodiments,the Myc peptide is a TAT-Myc peptide as described herein.

In some embodiments, a method described herein comprises introducing (orotherwise providing to) the Myc peptide into the anergic or tolerantB-cell ex vivo. In some embodiments, the Myc peptide is a fusionpeptide. In some embodiments, the Myc peptide comprises a PTD. In someembodiments, the Myc peptide is a TAT-Myc peptide as described herein.In some embodiments, the Myc peptide is a Myc-ER peptide as describedherein. In some embodiments, the Myc peptide is a Myc-GR peptide asdescribed herein.

In some embodiments, the anergic or tolerant B-cell is anergic ortolerant to the selected antigen prior to induction of Myc (e.g.,recombinant Myc) activity. In some embodiments, the selected antigen isa self-antigen.

In some embodiments, the nucleic acid sequence (e.g., a transgenicnucleic acid sequence) encoding the Myc peptide comprises a B-cellspecific promoter operably linked to the open reading frame encoding theMyc peptide. In some embodiments, the nucleic acid sequence (e.g., atransgenic nucleic acid sequence) encoding the Myc peptide comprises aninducible promoter operably linked to the open reading frame for the Mycpeptide. In some embodiments, the inducible promoter comprises one ormore TREs and the anergic or tolerant B-cell further expresses a tTApeptide or an rtTA peptide. In some embodiments, the anergic or tolerantB-cell expresses the tTA peptide.

In some embodiments, the Myc peptide is a Myc-ER fusion peptide. In someembodiments, inducing Myc (e.g., recombinant Myc) activity comprisescontacting the anergic or tolerant B-cell with an ER ligand. In someembodiments, the Myc peptide is a Myc-GR fusion peptide. In someembodiments, inducing Myc (e.g., recombinant Myc) activity comprisescontacting the anergic or tolerant B-cell with a GR ligand.

In some embodiments, the anergic or tolerant B-cell is a mouse cell. Insome embodiments, contacting the anergic or tolerant B-cell with anantigen comprises administering the selected antigen to an organism(i.e., immunizing or inoculating the organism). In some embodiments, theorganism is a xenomouse. In some embodiments, the immunized animal is anorganism (e.g., a mammal) carrying the nucleic acid sequence (e.g., atransgenic nucleic acid sequence) encoding the Myc peptide. In someembodiments, the nucleic acid sequence (e.g., a transgenic sequence) isan inducible nucleic acid sequence (e.g., a transgenic sequence). Insome embodiments, the inducible nucleic acid sequence (e.g., atransgenic sequence) comprises a promoter, and wherein the promotercomprises one or more TREs. In some embodiments, the organism (e.g., amammal) further carries and expresses a tTA nucleic acid sequence (e.g.,a transgenic sequence) or an rtTA nucleic acid sequence (e.g., atransgenic sequence). In some embodiments, the organism (e.g., a mammal)further carries and expresses a nucleic acid sequence (e.g., atransgenic sequence) encoding the selected antigen.

In some embodiment, the transformation of AN 1/T3 populations intoB-cell lines that produce antigen specific antibodies of interest occursin vivo. In some embodiments, a method described herein comprises theuse of MMTV-tTA/TRE-Myc organisms. In some embodiments, a methoddescribed herein comprises the use of Eu-Myc, Myc-GR, or Myc-ERorganisms. In some embodiments, any organism is used in which theorganism over-expresses Myc. In some embodiments, over-expression of MYCis induced by contacting the cell with a small molecule, a biologic, apeptide, an antibody, or a combination thereof. In some embodiments, thesmall molecule is an antagonist of Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, the small molecule is an antagonist ofMax-1. In some embodiments, the small molecule is an antagonist ofMxi-1. In some embodiments, the small molecule is an antagonist of MAD.In some embodiments, over-expression of MYC is induced by contacting thecell with a siRNA molecule for Max-1, Mxi-1, MAD, or a combinationthereof. In some embodiments, a method described herein comprises theuse of any organism in which Myc (e.g., recombinant Myc) activity isinducible. In some embodiments, a method described herein comprises theuse of any mouse in which Myc (e.g., recombinant Myc) activity ispresent or is inducible. In some embodiments, the organisms thatover-express MYC are mice. In some embodiments, the organism is axenomouse. In some embodiments, the organisms are exposed to theselected antigen and the organisms are maintained under condition inwhich MYC is over-ecpressed. In some embodiments, a method describedherein comprises organisms that express the selected antigen by means ofan integrated nucleic acid sequence (e.g., a transgenic sequence). Insome embodiments, tolerance to an antigen is broken by over-expressionof MYC in vivo. In some embodiments, B-cell tolerance to an antigen isbroken by Myc (e.g., recombinant Myc) activity in an AN1-T3 cell thatbinds the selected antigen. In some embodiments, tolerance is broken byinducing Myc (e.g., recombinant Myc) activity in an anergic B-cell invivo. In some embodiments, over-expression of MYC in vivo results in theexpansion of populations of B-cells, once anergic, that produceantibodies to the selected antigen. In some embodiments, MYC isover-ecpressed at 3 weeks of age or later to induce proliferation ofantigen-specific B-cells. In some embodiments, MYC is over-ecpressedfrom 3-30 weeks of age. In some embodiments, MYC is over-ecpressed from3-6, 3-12, or 3-24 weeks of age. In some embodiments, MYC isover-ecpressed at 6 weeks of age. In some embodiments, Myc is expressedat any point in the life cycle of the organism that provides for thegeneration of antigen-specific B-cells as described herein.

In some embodiments, MMTV-tTA/TRE-MYC mice exposed to antigen aremaintained on a doxycycline containing diet since conception. These miceare later switched to normal mouse chow to induce the over-expression ofMYC. In some embodiments, doxycycline is withdrawn from the diet at 3-30weeks of age. In some embodiments, doxycycline is withdrawn from thediet from 3-6, 3-12, or 3-24 weeks of age. In some embodiments,doxycycline is withdrawn from the diet at 6 weeks of age. In someembodiments, doxycycline is withdrawn from the diet at any point in thelife cycle of the organism that provides for the generation ofantigen-specific B-cells as described herein. The organisms are examineddaily for clinical signs associated with the development of B-celllymphomas. In some embodiments, mice that develop B-cell lymphomaspresent with scruffy fur, externally evident lymphadenopathy,dehydration, sluggishness, hind limb paralysis—ascending, etc. Oncetumors develop, leukocytes are isolated from the resulting tumors orfrom lymph nodes, spleen, bone marrow, or blood. In some embodiments,leukocytes are isolated prior to the development of tumors. In someembodiments, antigen-specific B-cells that over-express MYC arerecovered from the isolated leukocytes. In some embodiments, therecovered antigen-specific B-cells are expanded in vitro. In someembodiments, antigen specific antibody to antigens if recovered from theselected antigen-specific B-cells that are genetically altered toexpress Myc. In some embodiments, monoclonal cell lines are generatedfrom the recovered antigen-specific B-cells. In some embodiments,monoclonal antibodies are recovered from the selected antigen-specificB-cells. In some embodiments, the cells (e.g., mammalian cells) arestored frozen for future access to viable, primary antibody producingcells. In some embodiments, antibodies are isolated directly fromorganisms that present with B-cell lymphomas.

In some embodiments, provided herein is a method of producing anantibody specific for an antigen, comprising (a) providing an anergic ortolerant B-cell, wherein the anergic or tolerant B-cell comprises anucleic acid sequence (e.g., a transgenic sequence) encoding a Mycpeptide, (b) contacting the anergic or tolerant B-cell with an antigenin vivo wherein the anergic or tolerant B-cell binds specifically to theselected antigen; and (c) inducing Myc (e.g., recombinant Myc) activityin the anergic or tolerant B-cell.

In some embodiments, the Myc peptide is a fusion peptide. In someembodiments, the Myc peptide comprises a PTD. In some embodiments, theMyc peptide is a TAT-Myc peptide as described herein. In someembodiments, the Myc peptide is a Myc-ER peptide as described herein. Insome embodiments, the Myc peptide is a Myc-GR peptide as describedherein.

In some embodiments, the anergic or tolerant B-cells, upon exposure toantigen and Myc (e.g., recombinant Myc) activity, are transformed intoimmortal B-cells that produce an antibody that specifically binds to theselected antigen. In some embodiments, the anergic or tolerant B-cells,upon exposure to antigen and Myc (e.g., recombinant Myc) activity, aretransformed into immortal B-cells that produce an antibody thatspecifically binds to the selected antigen and cell fusion is notrequired.

In some embodiments, a method described herein further comprisesrecovering the antibody that specifically binds to the selected antigenfrom a plurality of the immortal B-cells that produce an antibody thatspecifically binds to the selected antigen. In some embodiments, theanergic or tolerant B-cell is present in an organism (e.g., a mammal)and inducing of Myc (e.g., recombinant Myc) activity in the anergic ortolerant B-cell occurs in vivo.

In some embodiments, a method described herein further comprisesintroducing (or otherwise providing to) a Myc peptide or a nucleic acidsequence (e.g., a transgenic sequence) encoding the Myc peptide into theanergic or tolerant B-cell ex vivo prior to the inducing step. In someembodiments, the Myc peptide is a fusion peptide. In some embodiments,the Myc peptide comprises a PTD. In some embodiments, the Myc peptide isa TAT-Myc peptide as described herein. In some embodiments, the Mycpeptide is a Myc-ER peptide as described herein. In some embodiments,the Myc peptide is a Myc-GR peptide as described herein.

In some embodiments, a method described herein comprises introducing (orotherwise providing to) the Myc peptide (e.g., a recombinant Myc peptide(e.g., a Myc fusion peptide as described herein)) into the anergic ortolerant B-cell ex vivo. In some embodiments, the Myc peptide is afusion peptide. In some embodiments, the Myc peptide comprises a PTD. Insome embodiments, the Myc peptide is a TAT-Myc peptide as describedherein. In some embodiments, the Myc peptide is a Myc-ER peptide asdescribed herein. In some embodiments, the Myc peptide is a Myc-GRpeptide as described herein.

In some embodiments, the anergic or tolerant B-cell is anergic ortolerant to the selected antigen prior to induction of Myc (e.g.,recombinant Myc) activity. In some embodiments, the selected antigen isa self-antigen. In some embodiments, the nucleic acid sequence (e.g., atransgenic nucleic acid sequence) encoding the Myc peptide comprises aB-cell specific promoter operably linked to the open reading frameencoding the Myc peptide. In some embodiments, the nucleic acid sequence(e.g., a transgenic nucleic acid sequence) encoding the Myc peptidecomprises an inducible promoter operably linked to the open readingframe for the Myc peptide. In some embodiments, the inducible promotercomprises one or more TREs and the recombinant anergic or tolerantB-cell further expresses a tTA peptide or an rtTA peptide. In someembodiments, the recombinant anergic or tolerant B-cell expresses thetTA peptide.

In some embodiments, the Myc peptide is a Myc-ER fusion peptide. In someembodiments, inducing Myc (e.g., recombinant Myc) activity comprisescontacting the recombinant anergic or tolerant B-cell with an ER ligand.In some embodiments, the Myc peptide is a Myc-GR fusion peptide. In someembodiments, inducing Myc (e.g., recombinant Myc) activity comprisescontacting the recombinant anergic or tolerant B-cell with a GR ligand.In some embodiments, the anergic or tolerant B-cell is a mouse cell. Insome embodiments, contacting the anergic or tolerant B-cell with anantigen comprises immunizing an organism with the selected antigen. Insome embodiments, the organism is a xenomouse. In some embodiments, theimmunized animal is an organism (e.g., a mammal) carrying the nucleicacid sequence (e.g., a transgenic nucleic acid sequence) encoding theMyc peptide. In some embodiments, the nucleic acid sequence (e.g., atransgenic sequence) is an inducible nucleic acid sequence (e.g., atransgenic sequence). In some embodiments, the inducible nucleic acidsequence (e.g., a transgenic sequence) comprises a promoter, and whereinthe promoter comprises one or more TREs. In some embodiments, theorganism (e.g., a mammal) further carries and expresses a tTA nucleicacid sequence (e.g., a transgenic sequence) or an rtTA nucleic acidsequence (e.g., a transgenic sequence). In some embodiments, theorganism (e.g., a mammal) further carries and expresses a nucleic acidsequence (e.g., a transgenic sequence) encoding the selected antigen.

In some embodiments, provided herein is a method of producing anantibody specific for an antigen, comprising (a) providing a CD79positive cell, wherein the CD79 positive cell comprises a nucleic acidsequence (e.g., a transgenic sequence) encoding a Myc peptide, (b)contacting the CD79 positive cell with an antigen wherein the CD79positive cell binds specifically to the selected antigen; and (c)inducing Myc (e.g., recombinant Myc) activity in the CD79 positive cell.

In some embodiments, the CD79 positive cell, upon exposure to antigenand Myc (e.g., recombinant Myc) activity, is transformed into animmortal CD79 positive cell that produce antibody that specificallybinds to the selected antigen. In some embodiments, the CD79 positivecells, upon exposure to antigen and Myc (e.g., recombinant Myc)activity, are transformed into immortal B-cells that produce antibodythat specifically binds to the selected antigen and cell fusion is notrequired. In some embodiments, a method described herein furthercomprises recovering the antibody that specifically binds to theselected antigen from a plurality of the immortal CD79 positive cellsthat produce antibody that specifically binds to the selected antigen.

In some embodiments, the CD79 positive cell is present in an organism(e.g., a mammal) and induction of Myc (e.g., recombinant Myc) activityin the CD79 positive cell occurs in vivo. In some embodiments, the Mycpeptide is a fusion peptide. In some embodiments, the Myc peptidecomprises a PTD. In some embodiments, the Myc peptide is a TAT-Mycpeptide as described herein. In some embodiments, the Myc peptide is aMyc-ER peptide as described herein. In some embodiments, the Myc peptideis a Myc-GR peptide as described herein.

In some embodiments, a method described herein further comprisesintroducing (or otherwise providing to) a Myc peptide or a nucleic acidsequence (e.g., a transgenic sequence) encoding the Myc peptide inot theCD79 positive cell ex vivo prior to the inducing step. In someembodiments, the Myc peptide is a fusion peptide. In some embodiments,the Myc peptide comprises a PTD. In some embodiments, the Myc peptide isa TAT-Myc peptide as described herein. In some embodiments, the Mycpeptide is a Myc-ER peptide as described herein. In some embodiments,the Myc peptide is a Myc-GR peptide as described herein.

In some embodiments, a method described herein comprises introducing (orotherwise providing to) the Myc peptide (e.g., a recombinant Myc peptide(e.g., a Myc fusion peptide as described herein)) into the CD79 positivecell ex vivo. In some embodiments, the Myc peptide is a fusion peptide.In some embodiments, the Myc peptide comprises a PTD. In someembodiments, the Myc peptide is a TAT-Myc peptide as described herein.In some embodiments, the Myc peptide is a Myc-ER peptide as describedherein. In some embodiments, the Myc peptide is a Myc-GR peptide asdescribed herein.

In some embodiments, the Myc peptide comprises a protein transductiondomain. In some embodiments, the CD79 positive cell is anergic ortolerant to the selected antigen prior to induction of Myc (e.g.,recombinant Myc) activity. In some embodiments, the selected antigen isa self-antigen. In some embodiments, the nucleic acid sequence (e.g., atransgenic nucleic acid sequence) encoding the Myc peptide comprises aB-cell specific promoter operably linked to the open reading frameencoding the Myc peptide. In some embodiments, the nucleic acid sequence(e.g., a transgenic nucleic acid sequence) encoding the Myc peptidecomprises an inducible promoter operably linked to the open readingframe for the Myc peptide. In some embodiments, the inducible promotercomprises one or more TREs and the recombinant CD79 positive cellfurther expresses a tTA peptide or an rtTA peptide. In some embodiments,the recombinant CD79 positive cell expresses the tTA peptide.

In some embodiments, the Myc peptide is a Myc-ER fusion peptide. In someembodiments, inducing Myc (e.g., recombinant Myc) activity comprisescontacting the recombinant CD79 positive cell with an ER ligand. In someembodiments, the Myc peptide is a Myc-GR fusion peptide. In someembodiments, inducing Myc (e.g., recombinant Myc) activity comprisescontacting the recombinant CD79 positive cell with a GR ligand. In someembodiments, the CD79 positive cell is a mouse cell. In someembodiments, contacting the CD79 positive cell with an antigen comprisesimmunizing an organism with the selected antigen. In some embodiments,the organism is a xenomouse. In some embodiments, the immunized animalis an organism (e.g., a mammal) carrying the nucleic acid sequence(e.g., a transgenic nucleic acid sequence) encoding the Myc peptide. Insome embodiments, the nucleic acid sequence (e.g., a transgenicsequence) is an inducible nucleic acid sequence (e.g., a transgenicsequence). In some embodiments, the inducible nucleic acid sequence(e.g., a transgenic sequence) comprises a promoter, and wherein thepromoter comprises one or more TREs. In some embodiments, the organism(e.g., a mammal) further carries and expresses a tTA nucleic acidsequence (e.g., a transgenic sequence) or an rtTA nucleic acid sequence(e.g., a transgenic sequence). In some embodiments, the organism (e.g.,a mammal) further carries and expresses a nucleic acid sequence (e.g., atransgenic sequence) encoding the selected antigen.

In some embodiments, immortal antibody producing cells are obtained fromany animal capable of generating B-cells, including but not limited tohumans, non-human primates, mice, rats, chickens, rabbits, pigs, cows orsheep.

In some embodiments, the antibody producing cells (e.g., animal B-cells)that are generated are immortal and/or malignant. Hybridomas that resultfrom the cell (e.g., a mammalian cell) fusion of a B-cell and a myelomapartner are typically heterokaryons and comprise two or more separatenuclei. In some embodiments, antibody producing cells (e.g., B-cells)have one nucleus. Hybridomas that result from the cell (e.g., amammalian cell) fusion of a B-cell and a myeloma partner contain anincreased number of chromosomes that often equals the sum of the numberof chromosomes present in each of the parent donor cells. Over time andover multiple cell passages, a hybridoma often loses some chromosomesbut generally retains more chromosomes than were present in any one ofthe donor parent cells. In some embodiments, the antibody producingcells (e.g., B-cells), that are generated by any of the methodsdescribed herein, display the same number of chromosomes as were presentbefore Myc was over-expressed. In some embodiments, the antibodyproducing cells that are generated by the methods described hereincomprise no more than 5 additional chromosomes when compared to a timejust prior to antibody production. In some embodiments, the antibodyproducing cells that are generated by the methods described hereincomprise no more than 4 additional chromosomes when compared to a timejust prior to antibody production. In some embodiments, the antibodyproducing cells that are generated by the methods described hereincomprise no more than 3 additional chromosomes when compared to a timejust prior to antibody production. In some embodiments, the antibodyproducing cells that are generated by the methods described hereincomprise no more than 2 additional chromosomes when compared to a timejust prior to antibody production. In some embodiments, the antibodyproducing cells that are generated by the methods described hereincomprise no more than 1 additional chromosome when compared to a timejust prior to antibody production. In some embodiments, the antibodyproducing cells that are generated by the methods described hereincomprise no additional chromosomes when compared to a time just prior toantibody production.

In some embodiments, reactive splenic B-cells are cultured in thepresence of an onco-peptide (e.g., Myc). In some embodiments, reactivesplenic B-cells are cultured in the presence of a Myc peptide. In someembodiments, reactive splenic B-cells are cultured in the presence of aTAT-Myc fusion peptide disclosed herein. In some embodiments, culturingreactive splenic B-cells in the presence of a TAT-Myc fusion peptidefacilitates the fusion of the B-cell and a myeloma fusion partner. Insome embodiments, culturing reactive splenic B-cells in the presence ofa TAT-Myc fusion peptide ameliorates the need for using reactive B-cellsin S-phase when fusing a B-cell with a myeloma fusion partner.

TAT-MYC Fusion Peptide

Disclosed herein, in certain embodiments, is a fusion peptide comprising(a) a transporter peptide sequence; and (b) a MYC sequence. In someembodiments, the fusion peptide is a peptide of Formula (I): transporterpeptide sequence-MYC sequence.

In some embodiments, a fusion peptide disclosed herein comprises (a) atransporter peptide sequence; (b) a MYC sequence; and (c) one or moremolecules that link the transporter peptide sequence and the MYCsequence. In some embodiments, the fusion peptide is a peptide ofFormula (II):

transporter peptide sequence-X-MYC sequence,

wherein -X- is molecule that links the transporter peptide sequence andthe MYC sequence. In some embodiments, -X- is an amino acid. In someembodiments, -X- is at least one amino acid.

In some embodiments, a fusion peptide disclosed herein comprises (a)TAT, and (b) c-MYC. In some embodiments, a fusion peptide disclosedherein comprises (a) TAT_([48-57]), and (b) c-MYC. In some embodiments,a fusion peptide disclosed herein comprises (a) TAT_([57-48]), and (b)c-MYC.

In some embodiments, a fusion peptide disclosed herein comprises (a)TAT, (b) a linker amino acid, and (c) c-MYC. In some embodiments, afusion peptide disclosed herein comprises (a) TAT_([48-57]), (b) alinker amino acid, and (c) c-MYC. In some embodiments, a fusion peptidedisclosed herein comprises (a) TAT_([57-48]), (b) a linker amino acid,and (c) c-MYC.

In some embodiments, a fusion peptide disclosed herein further comprisesat least one amino acid sequence that facilitates purification of thefusion protein. In some embodiments, a fusion peptide disclosed hereincomprises a protein tag. In some embodiments, a fusion peptide disclosedherein comprises a polyhistidine tag. In some embodiments, a fusionpeptide disclosed herein comprises an epitope tag. In some embodiments,a fusion peptide disclosed herein comprises at least one of apolyhistidine tag and an epitope tag. In some embodiments, a fusionpeptide disclosed herein comprises at least one of a 6-histidine tag(SEQ ID NO. 2) and a V5 epitope tag.

In some embodiments, the histidine tag is a 6-histidine tag (SEQ ID NO.2). In some embodiments, the histidine tag comprises the sequence HHHHHH(SEQ ID NO. 2). In some embodiments, a polyHis tag is added to a fusionprotein disclosed herein by any suitable method. In some embodiments, aTAT-MYC peptide sequence is cloned into an expression vector encoding apolyHis-tag. In some embodiments, a polyHis tag is added by PCR (i.e.,the PCR primers comprise a polyHis sequence).

In some embodiments, a fusion peptide disclosed herein further comprisesat least one protein tag. In some embodiments, a fusion peptidedisclosed herein comprises an epitope tag. In some embodiments, a fusionpeptide disclosed herein further comprises a V5 epitope tag. In someembodiments, the V5 tag comprises the amino acids: GKPIPNPLLGLDST (SEQID NO. 3). In some embodiments, the V5 tag comprises the amino acids:IPNPLLGLD (SEQ ID NO. 4). In some embodiments, a V5 tag is added to afusion protein disclosed herein by any suitable method. In someembodiments, a TAT-MYC peptide sequence is cloned into an expressionvector encoding a V5 tag. In some embodiments, a V5 tag is added by PCR(i.e., the PCR primers comprise a V5 sequence).

In some embodiments, the amino acids are in the D formation. In someembodiments, the amino acids are in the L formation. In someembodiments, a first plurality of amino acids are in the D formation anda second plurality are in the L formation.

In some embodiments, the MYC Increasing Agent comprises:

(SEQ ID NO. 5) MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSP SYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDE TFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCST SSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAK RVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRS FFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH

Construction of a TAT-MYC Peptide

In some embodiments, a TAT-MYC fusion peptide disclosed herein isconstructed by any suitable method. In some embodiments, a nucleotidesequence encoding a TAT-MYC fusion peptide is generated by PCR. In someembodiments, a forward primer for a human MYC sequence comprises an inframe N-terminal 9-amino-acid sequence of the TAT protein transductiondomain (i.e., RKKRRQRRR; (SEQ ID NO. 5)). In some embodiments, a reverseprimer for a human MYC sequence is designed to remove the stop codon. Insome embodiments, the PCR product is cloned into any suitable expressionvector (hereinafter, p-TAT-MYC). In some embodiments, the expressionvector comprises a polyhistidine tag and a V5 tag.

Recombinant Polypeptides

Described herein are certain recombinant polypeptides. In certaininstances, antibodies comprise light and a heavy polypeptide chains. Insome embodiments, DNA sequences that encode the light and the heavychains of the antibody are made, either simultaneously or separately,using reverse transcriptase and DNA polymerase in accordance withsuitable methods. In some embodiments, PCR is initiated by consensusconstant region primers or by more specific primers based on thepublished heavy and light chain DNA and amino acid sequences. Asdiscussed above, in some embodiments, PCR is also used to isolate DNAclones encoding the antibody light and heavy chains. In someembodiments, the libraries are screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

In some embodiments, DNA, e.g., plasmid DNA, is isolated from the cell(e.g., a mammalian cell) by any suitable method, restriction mapped andsequenced in accordance with any suitable techniques set forth indetail, e.g., in the foregoing references relating to recombinant DNAtechniques. In some embodiments, the DNA is synthetic at any pointduring the isolation process or subsequent analysis.

In some embodiments, following manipulation of the isolated geneticmaterial to provide antibodies, or antigen-binding fragments, variants,or derivatives thereof, the polynucleotides encoding the antibodies areinserted in an expression vector for introduction into host cells whichare therein used to produce the desired quantity of antibody.

In certain embodiments, recombinant expression of an antibody, orfragment, derivative or analog thereof, e.g., a heavy or light chain ofan antibody that binds to a target molecule utilizes construction of anexpression vector containing a nucleic acid that encodes the antibody.Once a polynucleotide encoding an antibody molecule or a heavy or lightchain of an antibody, or portion thereof (preferably containing theheavy or light chain variable domain), is obtained, the vector for theproduction of the antibody molecule is in some embodiments, produced byrecombinant DNA technology using any suitable techniques. Thus,processes for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. In some embodiments, processes any suitable manner of constructexpression vectors containing antibody coding sequences comprisingappropriate transcriptional and translational control signals isutilized in the a method disclosed herein. These processes include, forexample, in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Thus, in some embodiments, providedherein are replicable vectors comprising a nucleotide sequence encodingan antibody molecule, or a heavy or light chain thereof, or a heavy orlight chain variable domain, operably linked to a promoter. In someembodiments, vectors comprise the nucleotide sequence encoding theconstant region of the antibody and the variable domain of the antibodyis incorporated into the vector for expression of the entire heavy orlight chain.

In some embodiments, the cloned variable region genes are inserted intoan expression vector along with the heavy and light chain constantregion genes synthesized as discussed above. In some embodiments, anexpression vector that is capable of eliciting expression in eukaryoticcells is used. Examples of suitable vectors include, but are not limitedto plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2,pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (availablefrom Invitrogen, San Diego, Calif.), and plasmid pCI (available fromPromega, Madison, Wis.). In certain instances, screening large numbersof transformed cells for those that express suitably high levels ofimmunoglobulin heavy and light chains is accomplished by any suitablemethod, for example, by robotic systems.

In some embodiments, once the vector or DNA sequence encoding anantibody is prepared, the expression vector is introduced into anappropriate host cell as disclosed herein. Introduction of the plasmidinto the host cell is accomplished by any suitable method. Theseinclude, but are not limited to, transfection (includingelectroporation), protoplast fusion, calcium phosphate precipitation,cell fusion with enveloped DNA, DEAE-dextran mediated transfection,microinjection, cationic lipid-mediated transfection, transduction,scrape loading, ballistic introduction and infection with intact virus.See, e.g., Ridgway (1988) “Mammalian Expression Vectors” in Vectors, ed.Rodriguez and Denhardt (Butterworths, Boston, Mass.), Chapter 24.2, pp.470-472; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.In certain instances, host cells harboring the expression construct aregrown under conditions appropriate to the production of the light chainsand heavy chains, and assayed for heavy and/or light chain proteinsynthesis. Exemplary assay techniques include ELISA, radioimmunoassay(RIA), or fluorescence-activated cell sorter analysis (FACS),immunohistochemistry and the like.

As disclosed herein, introducing (or otherwise providing to) nucleicacids and/or vectors into host organisms are achieved by any suitablemethod. Examples of such processes include but are not limited to genegun techniques, naked DNA immunization, the development of transgenic ortranschromosomal organisms, lentiviral mediated production of transgenicorganisms, vaccinia viruses transduction and adenovirus.

As disclosed herein, introducing (or otherwise providing to) antigensinto organisms or cells comprises any manner suitable. Some examples ofsuch processes include but are not limited to the use of Tat fusionpeptides, various immunization protocols, infection by virus, andintroduction of whole cells expressing antigen.

In some embodiments, an expression vector is transferred to a host cellby any suitable method, and the transfected cells are then cultured byconventional techniques or techniques disclosed herein to produce anantibody for use in the methods described herein. Thus, provided incertain embodiments, herein are host cells that contain a polynucleotideencoding an antibody, or a heavy or light chain thereof, operably linkedto a heterologous promoter. In some embodiments, for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains are used to express of the entire immunoglobulin molecule.

In some embodiments, a number of selection systems are used including,but not limited to, the herpes simplex virus thymidine kinase (Wigler etal. (1977) Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski (1992) Proc. Natl. Acad. Sci. USA 48:202), andadenine phosphoribosyltransferase (Lowy et al. (1980) Cell 22:817) genesemployed in tk-, hgprt- or aprt-cells, respectively. Also, in someembodiments, antimetabolite resistance is used as the basis of selectionfor the following genes: dhfr, which confers resistance to methotrexate(Wigler et al. (1980) Natl. Acad. Sci. USA 77:357; O'Hare et al. (1981)Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95;Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan(1993) Science 260:926-932; and Morgan and Anderson (1993) Ann. Rev.Biochem. 62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); andhygro, which confers resistance to hygromycin (Santerre et al. (1984)Gene 30:147. In some embodiments, methods commonly known in the art ofrecombinant DNA technology which are used as described in Ausubel et al.(1993) Current Protocols in Molecular Biology (John Wiley & Sons, NY);Kriegler (1990) “Gene Transfer and Expression” in A Laboratory Manual(Stockton Press, NY); Dracopoli et al. (eds) (1994) Current Protocols inHuman Genetics (John Wiley & Sons, N.Y.) Chapters 12 and 13;Colberre-Garapin et al. (1981) J. Mol. Biol. 150:1.

In some embodiments, the expression level of an antibody molecule isincreased by vector amplification (for a review, see Bebbington andHentschel (1987) “The Use of Vectors Based on Gene Amplification for theExpression of Cloned Genes in Mammalian Cells in DNA Cloning” (AcademicPress, NY) Vol. 3. When a marker in the vector system expressingantibody is amplifiable, an increase in the level of inhibitor presentin the culture of the host cell will increase the number of copies ofthe marker gene. Since the amplified region is associated with theantibody gene, production of the antibody will also increase (Crouse etal. (1983) Mol. Cell. Biol. 3:257).

Harvesting Antibodies

In various embodiments, of any of the methods disclosed herein,antibodies that specifically binds to the selected antigen, as well asthe cell (e.g., a mammalian cell) producing such antibodies, areisolated (i.e., harvested) directly from the tissues (spleen, lymphnodes, blood, etc.) or serum of an organism (e.g., mouse, rabbit). Insome embodiments, the antibodies are isolated from splenic tissue. Insome embodiments, the antibodies are harvested from lymph nodes. In someembodiments, the antibodies are harvested from blood. In someembodiments, the tissue is harvested by any suitable method.

In some embodiments, the antibodies are harvested in the presence of theselected antigen. In some embodiments, the antibodies are harvested inthe presence of an onco-peptide (e.g., Myc, TAT-Myc). In someembodiments, the antibodies are harvested in the presence of anonco-peptide (e.g., Myc, TAT-Myc) and the selected antigen. In someembodiments, the antibodies are harvested in the presence of a Mycpeptide. In some embodiments, the antibodies are harvested in thepresence of a TAT-Myc fusion peptide disclosed herein.

In some embodiments, harvesting antibodies in the presence of theselected antigen, the selected antigen and an onco-peptide, the selectedantigen and Myc, or the selected antigen and TAT-Myc reduces theconcentration of tolerant and/or anergic cells. In some embodiments,harvesting antibodies in the presence of the selected antigen andTAT-Myc reduces the concentration of tolerant and/or anergic cells(e.g., B-cells).

Scale Up

In some embodiments, antibodies produced by a method disclosed hereinare reengineered for large scale production. In some embodiments,antibodies produced by a method disclosed herein are reengineered forlarge scale production for clinical use or for use in a diagnosticassay, e.g., under GMP or cGMP guidelines. In some embodiments, aselected antibody is prepared by:

-   -   a. recovering a nucleic acid encoding the antibody from cell        (e.g., a B-cell) that comprises a nucleic acid sequence (e.g., a        transgenic sequence) encoding an onco-peptide (e.g., recombinant        onco-peptide; e.g., Myc) and/or a recombinant onco-peptide        (e.g., recombinant onco-peptide; e.g., Myc);    -   b. introducing (or otherwise providing to) the nucleic acid        encoding the antibody into a cell that is suitable for large        scale production of an antibody.

In some embodiments, cell that is genetically altered to express anonco-peptide and comprises a nucleic acid encoding the antibody isprepared according to any process described herein. In certainembodiments, the method further comprises selecting an antibody. In someembodiments, the antibody is a human antibody. In certain embodiments,cell expresses human antibodies. In certain embodiments, the methodfurther comprises contacting cell (e.g., B-cell) that comprises anucleic acid sequence (e.g., a transgenic sequence) encoding anonco-peptide (e.g., recombinant onco-peptide; e.g., Myc) with a selectedantibody.

In some embodiments, the method further comprises culturing the cell(e.g., a mammalian cell) that is suitable for large scale production ofan antibody under conditions suitable for production of the antibody. Incertain embodiments, the method further comprises recovering a quantityof the antibody, e.g., at least 0.1 kg, at least 0.5 kg, at least 1 kg,at least 10 kg, or at least 100 kg.

Any suitable cell line for large scale production is used in suchprocesses. Examples of cells or cell lines that are suitable for largescale production include, but are not limited to, Chinese hamster ovaryCHO, NSO, BSC-1 cells, LLC-MK cells, CV-1, HeLa, HEK293, VERO, MDBK,MDCK, MDOK, CRFK, RAF, TCMK, LLC-PK, PK15, WI-38, MRC-5, T-FLY, BHK, BRL3A, HepG2, HT1080 or derivatives thereof.

In some embodiments, nucleic acids encoding an antibody, produced by themethods described herein, are recovered from a B-cell that isgenetically altered to express Myc and are reintroduced into a CHO cellor derivatives thereof (including dhfr-CHO cells used with a DHFRselectable marker), for the purpose of large scale production of theantibody. In some embodiments, nucleic acids encoding the antibody ofinterest are recovered from a B-cell that is genetically altered toexpress Myc and are reintroduced into a plant cell that is suitable forlarge scale production of an antibody. In some embodiments, nucleicacids encoding an antibody, produced by the methods described herein,are recovered from a B-cell that is genetically altered to express Mycand are reintroduced into a plant, part of a plant or a plant seed thatis suitable for large scale production of an antibody. In someembodiments, nucleic acids encoding an antibody, produced by the methodsdescribed herein, are recovered from a B-cell that is geneticallyaltered to express Myc and are reintroduced into cotton, potato,soybean, corn, maize, tobacco, soybean, alfalfa, or rice for the purposeof large scale production of the antibody. In some embodiments, nucleicacids encoding an antibody, produced by the methods described herein,are recovered from a B-cell that is genetically altered to express Mycand are reintroduced into a yeast for the purpose of large scaleproduction of the antibody. Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220 and R. J. Kaufman and P. A. Sharp (1982) Mol.Biol. 159:601-621.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for cell (e.g., a mammalian cell) cultivationunder tissue culture conditions include homogeneous suspension culture,e.g. in an airlift reactor or in a continuous stirrer reactor, orimmobilized or entrapped cell culture, e.g. in hollow fibers,microcapsules, on agarose microbeads or ceramic cartridges. If necessaryand/or desired, in some embodiments, solutions of polypeptides arepurified in any manner suitable. Examples of such methods include butare not limited to chromatography methods, for example gel filtration,ion-exchange chromatography, chromatography over DEAE-cellulose or(immuno-) affinity chromatography.

Once an antibody molecule is recombinantly expressed, in someembodiments, the antibody is purified by any suitable method forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, protein A affinity,protein G affinity, gel filtration and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of antibodies. In some embodiments,isolated antibodies include serum containing such antibodies, orantibodies that have been purified to varying degrees.

Antigens

In some embodiments, the methods disclosed herein are used to generateantibodies that bind and/or are specific to antigens. In someembodiments, the selected antigens are therapeutic targets. In someembodiments, the antibodies are generated to bind an antigen wherein theselected antigen is a TNF or TNF receptor family member or TNF likemolecule. In some embodiments, the TNF or TNF receptor family member orTNF like molecule is selected from the list: TNF-alpha, lymphotoxin(LT), LT-alpha (also known as TNF-beta), LT-beta (found in complexheterotrimers with LT-alpha), OPGL, FasL, CD27L, CD3OL, CD40L, 4-1BBL,DcR3, OX40, OX40L, TNF-gamma (International Publication No. WO96/14328), TRAIL, AIM-II (International Publication No. WO 97/34911),APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha (InternationalPublication No. WO 98/07880), TR6 (International Publication No. WO98/30694), OPG, and neutrokine-alpha (International Publication No. WO98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas,CD30, CD27, CD40 and 4-1BB, TR2 (International Publication No. WO96/34095), DR3 (International Publication No. WO 97/35904), TR5(International Publication No. WO 98/30693), TR6 (InternationalPublication No. WO 98/30694), TR7 (International Publication No. WO98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10(International Publication No. WO 98/54202), 312C2 (InternationalPublication No. WO 98/06842), and TR12, LIGHT, TRANCE, TACI, BAFFR,BCMA, NGFR, APRIL, CD154, CD70, CD153 including all soluble and membranebound forms of these molecules. In some embodiments, the antibodies aregenerated to bind an antigen wherein the selected antigen is anycytokine or interleukin. In some embodiments, the cytokine orinterleukin antigen is selected from the list: IL-1alpha, IL-1beta,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,IFN-alpha, IFN-beta, IFN-gamma, MIF, GM-CSF, and G-CSF. In someembodiments, the antibodies are generated to bind an antigen wherein theselected antigen is chemokine or chemokine receptor. In someembodiments, the chemokine antigen is selected from the list:gamma-interferon inducible protein-10 (γIP-10), interleukin-8 (IL-8),platelet factor-4 (PF4), neutrophil activating protein (NAP-2), GRO-α,GRO-β, GRO-γ, neutrophil-activating peptide (ENA-78), granulocytechemoattractant protein-2 (GCP-2), and stromal cell-derived factor-1(SDF-1, or pre-B cell stimulatory factor (PBSF)); and/or β (CC)chemokine selected from the group consisting of: RANTES (regulated onactivation, normal T expressed and secreted), macrophage inflammatoryprotein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta(MIP-1β), monocyte chemotactic protein-1 (MCP-1), monocyte chemotacticprotein-2 (MCP-2), monocyte chemotactic protein-3 (MCP-3), monocytechemotactic protein-4 (MCP-4) macrophage inflammatory protein-1 gamma(MIP-1γ), macrophage inflammatory protein-3 alpha (MIP-3α), macrophageinflammatory protein-3 beta (MIP-3β), macrophage inflammatory protein-4(MIP-4/DC-CK-1/PARC), eotaxin, Exodus, and 1-309; and/or the γ(C)chemokine, and lymphotactin. In some embodiment the selected antigen isa chemokine receptor. In some embodiments, the chemokine ligand isselected from the list comprising CCL1, CCL2, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL2, CCL14, CCL15, CCL16,CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26,CCL27 and CCL28. In some embodiments, the selected antigen is achemokine receptor selected from the list: CXCR1, CXCR2, CXCR3, CXCR4,CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9, CCR10, and CCR11. In some embodiments, the selected antigen is afibroblast growth factor. In some embodiments, the fibroblast growthfactor is selected from the list: FGF-1, FGF-2, FGF-3, FGF-4, FGF-5,FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, andFGF-15. In some embodiments, the selected antigen is a cell-surfaceprotein. In some embodiments, the cell (e.g., a mammalian cell)-surfaceprotein is a human leukocyte differentiation antigen. In someembodiments, the human leukocyte differentiation antigen is selectedfrom the list: CD1, CD2, CD3, CD4, CD8, CD10, CD20, CD100, CD280, CD281,CD282, CD283, CD284, and CD289. In some embodiments, the human leukocytedifferentiation antigen is selected from the list comprising CD1 thoughCD300. In some embodiments, the selected antigen is an intracellularcancer biomarker. In some embodiments, the selected antigen is encodedby HIV. In some embodiments, the selected antigen is a polypeptidecomprising all or a portion of an HIV encoded polypeptide selected fromthe list: Gag, pol, protease (prot), reverse transcriptase, integrase,RNAseH, Tat, Rev, Nef, Vpr, Vpu, Vif and Env (e.g., gp120, gp140, gp41and gp160). In some embodiments, the selected antigen is an antigenderived from a species of Orthomyxoviridae. In some embodiments, theselected antigen derived from Othomyxoviridae is derived from a strainof influenza A selected from, by way of non-limiting example, H1N1,H2N2, H3N2, H5N1, H7N7, H1N2 H9N2, H7N2, H7N3, and H10N7. In someembodiments, the selected antigen is a hemagglutinin glycoproteinderived from Othomyxoviridae. In some embodiments, the selected antigenis a neuraminidase derived from Othomyxoviridae. In some embodiments,the selected antigen is derived from a coronavirus. In some embodiments,the selected antigen is derived from a SARS coronavirus.

In some embodiments, the methods disclosed herein are used to generateantibodies that bind and/or are specific to selected self antigens. Insome embodiment, the self antigens originate from within an organism,tissue, or cell. In some embodiments, a self antigen comprises anendogenous antigen. In some embodiments, a self antigen comprises anendogenous antigen produced by an endogenous retrovirus. In someembodiments, self antigens comprise neo-self antigens, microbially orparasite encoded neo-self antigens, or other neo-self antigens expressedas a result of genetic alteration to an organism or cell. In someembodiments, a chimeric mouse expresses a neo-self antigen. In someembodiments, the methods disclosed herein are used to generateantibodies that bind and/or are specific to selected self antigens thatantagonize a disease process. In some embodiments, the disease processis an autoimmune disorder. In some embodiments, the methods disclosedherein are used to generate antibodies that bind and/or are specific toselected auto antigens or immunologically reactive epitopes that mimicthat of a self antigen or autoantigen.

In some embodiments, the methods disclosed herein are used to generateantibodies that bind and/or are specific to antigens and antagonize adisease processes. In some embodiments, the selected antigens areantibodies that contribute to the pathogenesis of a disorder or diseaseprocess.

In some embodiments, utilizing the techniques disclosed herein,monoclonal antibodies displaying a multitude of specificities aregenerated that neutralize entire clades of HIV variants. In someembodiments, this is accomplished by targeting an obligatory structuralcomponent of the HIV viral envelope proteins. In some embodiments, thisis accomplished by targeting a host co-receptor protein.

In some embodiments, the selected antigen is an autoimmune antigen. Insome embodiments, the autoimmune antigen is selected from the list:thyroglobulin, thyroid peroxidase, cytoplasmic TSH receptor, intrinsicfactor, beta-adrenergic receptor, acetyl choline receptor, myelin basicprotein, amyloid beta, amyloid precursor protein, collagen,sodium-iodide symporter, histone polypeptides and nucleic acids.

The technologies disclosed herein provide new strategies for the rapiddevelopment of diagnostic and therapeutic antibodies for the detectionand treatment of emerging infectious diseases and chronic illnesses suchas cancer and autoimmunity. In some embodiments, the antibodies producedby the methods disclosed herein are used for detecting or quantitatingvarious serum proteins for diagnostic purposes. In some embodiments, theantibodies produced by the methods disclosed herein are used fordetecting or quantitating various markers for diagnostic purposes. Insome embodiments, the markers are cell surface markers. In someembodiments, the markers are cancer or tumor markers.

In some embodiments, the antibodies produced by the methods disclosedherein are used for therapeutic treatments. In some embodiments, theantibodies produced by the methods disclosed herein are reagents usedfor diagnostic and/or research purposes.

In some embodiments, antibodies capable of selectively binding to a widerange of antigens are produced by a method described herein. Any antigencapable of inducing an immune response when introduced to an organism issuitable for use in a method described herein. In some embodiments,antigens that are normally subjected to self tolerance mechanisms, suchas auto antigens, are used in a method described herein. In someembodiments, the selected antigens fail to induce antibodies utilizingstandard immunization protocols.

In some embodiments, a method described herein utilizes any organismsuitable for antibody production, including a cell or tissue thereof. Insome embodiments, organisms suitable for antibody production include anyanimal of the Vertebrate class, Mammalia (i.e., mammals), including, andwithout limitation, primates, rodents, livestock and domestic pets. Incertain instances, in the production of an antibody, a suitableexperimental animal, such as, for example, but not limited to, a rabbit,a sheep, a hamster, a guinea pig, a mouse, a rat, or a chicken, isadministered (i.e., immunized with, inoculated against) an antigenagainst which an antibody is desired.

Pharmaceutical Compositions and Methods of Administration

In certain embodiments, provided herein, are pharmaceutical compositionscomprising an antibody and one or more physiologically acceptablecarriers. Physiologically acceptable carriers include excipients andauxiliaries which facilitate processing of the active agents intopreparations which are used pharmaceutically. In certain instances,proper formulation is dependent upon the route of administration chosen.A summary of pharmaceutical compositions is found, for example, inRemington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton,Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975;Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms andDrug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins,1999).

Provided herein are pharmaceutical compositions that include an antibodyand a pharmaceutically acceptable diluent(s), excipient(s), orcarrier(s). In addition, an antibody is optionally administered aspharmaceutical compositions in which they are mixed with other activeingredients, as in combination therapy. In some embodiments, thepharmaceutical compositions includes other medicinal or pharmaceuticalagents, carriers, adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In addition, the pharmaceutical compositionsalso contain other therapeutically valuable substances.

A pharmaceutical composition refers to a mixture of an antibody withother chemical components, such as carriers, stabilizers, diluents,dispersing agents, suspending agents, thickening agents, and/orexcipients. The pharmaceutical composition facilitates administration ofan antibody to an organism. In practicing the methods of treatment oruse provided herein, therapeutically effective amounts of an antibodyare administered in a pharmaceutical composition to an organism (e.g., amammal) having a condition, disease, or disorder to be treated.Preferably, the organism (e.g., a mammal) is a human. A therapeuticallyeffective amount varies depending on the severity and stage of thecondition, the age and relative health of the subject, the potency ofthe antibody used and other factors. Antibodies are optionally usedsingly or in combination with one or more therapeutic agents ascomponents of mixtures.

The pharmaceutical formulations described herein are optionallyadministered to a subject by multiple administration routes, includingbut not limited to, oral, parenteral (e.g., intravenous, subcutaneous,intramuscular), intranasal, buccal, topical, rectal, or transdermaladministration routes. The pharmaceutical formulations described hereininclude, but are not limited to, aqueous liquid dispersions,self-emulsifying dispersions, solid solutions, liposomal dispersions,aerosols, solid dosage forms, powders, immediate release formulations,controlled release formulations, fast melt formulations, tablets,capsules, pills, delayed release formulations, extended releaseformulations, pulsatile release formulations, multiparticulateformulations, and mixed immediate and controlled release formulations.

Methods of Dosing and Treatment Regimens

An antibody is optionally used in the preparation of medicaments for theprophylactic and/or therapeutic treatment of inflammatory conditions orconditions that would benefit, at least in part, from amelioration. Inaddition, a method for treating any of the diseases or conditionsdescribed herein in a subject in need of such treatment, involvesadministration of pharmaceutical compositions containing an antibody asdescribed herein, or a pharmaceutically acceptable salt,pharmaceutically acceptable N-oxide, pharmaceutically active metabolite,pharmaceutically acceptable prodrug, or pharmaceutically acceptablesolvate thereof, in therapeutically effective amounts to said subject.

Furthermore, in some embodiments, provided herein is a method oftreating a disorder mediated by an antigen by administering atherapeutically effective antibody that binds or specifically binds tothe selected antigen to an individual in need thereof. Exemplaryantigens are set forth herein and include, by way of non-limitingexample, viral antigens (e.g., HIV antigens, influenza antigens, SARSantigens), autoantigens, tumor antigens, various pathogen relatedantigens and the like. Likewise, disorders mediated by the selectedantigen include any disorder so mediated, including, by way ofnon-limiting example, viral infections and syndromes caused thereby(e.g., HIV, influenza, and SARS), as well as pathogenic and parasiticinfections and syndromes caused thereby.

In certain instances wherein the patient's condition does not improve,upon the doctor's discretion the administration of an antibody isoptionally administered chronically, that is, for an extended period oftime, including throughout the duration of the patient's life in orderto ameliorate or otherwise control or limit the symptoms of thepatient's disease or condition.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of an antibody is optionally givencontinuously; alternatively, the dose of drug being administered istemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). The length of the drug holiday optionallyvaries between 2 days and 1 year, including by way of example only, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days,20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350days, or 365 days. The dose reduction during a drug holiday includesfrom 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of thesymptoms, to a level at which the improved disease, disorder orcondition is retained. In some embodiments, patients requireintermittent treatment on a long-term basis upon any recurrence ofsymptoms.

In some embodiments, the pharmaceutical composition described herein isin unit dosage forms suitable for single administration of precisedosages. In unit dosage form, the formulation is divided into unit dosescontaining appropriate quantities of an antibody. In some embodiments,the unit dosage is in the form of a package containing discretequantities of the formulation. Non-limiting examples are packagedtablets or capsules, and powders in vials or ampoules. In someembodiments, aqueous suspension compositions are packaged in single-dosenon-reclosable containers. Alternatively, multiple-dose reclosablecontainers are used, in which case it is typical to include apreservative in the composition. By way of example only, formulationsfor parenteral injection are presented in unit dosage form, whichinclude, but are not limited to ampoules, or in multi dose containers,with an added preservative.

The daily dosages appropriate for an antibody are from about 0.01 to 2.5mg/kg per body weight. An indicated daily dosage in the larger organism(e.g., a mammal), including, but not limited to, humans, is in the rangefrom about 0.5 mg to about 100 mg, conveniently administered in divideddoses, including, but not limited to, up to four times a day or inextended release form. Suitable unit dosage forms for oraladministration include from about 1 to 50 mg active ingredient. Theforegoing ranges are merely suggestive, as the number of variables inregard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesare optionally altered depending on a number of variables, not limitedto the activity of the antibody used, the disease or condition to betreated, the mode of administration, the requirements of the individualsubject, the severity of the disease or condition being treated, and thejudgment of the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental organisms,including, but not limited to, the determination of the LD50 (the doselethal to 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD50 and ED50. An antibody exhibiting high therapeuticindices is preferred. The data obtained from cell culture assays andanimal studies are optionally used in formulating a range of dosage foruse in human. The dosage of such an antibody lies preferably within arange of circulating concentrations that include the ED50 with minimaltoxicity. The dosage optionally varies within this range depending uponthe dosage form employed and the route of administration utilized.

Combination Treatments

Antibody compositions described herein are also optionally used incombination with other therapeutic reagents that are selected for theirtherapeutic value for the condition to be treated. In general, thecompositions described herein and, in embodiments, where combinationaltherapy is employed, other agents do not have to be administered in thesame pharmaceutical composition, and, because of different physical andchemical characteristics, are optionally administered by differentroutes. The initial administration is optionally made according toestablished protocols, and then, based upon the observed effects, thedosage, modes of administration and times of administration subsequentlymodified.

In certain instances, it is appropriate to administer an antibodycomposition as described herein in combination with another therapeuticagent. By way of example only, if one of the side effects experienced bya patient upon receiving an antibody composition as described herein isnausea, then it is appropriate to administer an anti-nausea agent incombination with the initial therapeutic agent. Or, by way of exampleonly, the therapeutic effectiveness of an antibody are enhanced byadministration of an adjuvant (i.e., by itself the adjuvant has minimaltherapeutic benefit, but in combination with another therapeutic agent,the overall therapeutic benefit to the patient is enhanced). Or, by wayof example only, the benefit experienced by a patient is increased byadministering an antibody with another therapeutic agent (which alsoincludes a therapeutic regimen) that also has therapeutic benefit. Inany case, regardless of the disease, disorder or condition beingtreated, the overall benefit experienced by the patient is either simplyadditive of the two therapeutic agents or the patient experiences asynergistic benefit.

Therapeutically-effective dosages vary when the drugs are used intreatment combinations. Methods for experimentally determiningtherapeutically-effective dosages of drugs and other agents for use incombination treatment regimens are documented methodologies. One exampleof such a method is the use of metronomic dosing, i.e., providing morefrequent, lower doses in order to minimize toxic side effects.Combination treatment further includes periodic treatments that startand stop at various times to assist with the clinical management of thepatient.

In any case, the multiple therapeutic agents (one of which is anantibody as described herein) are administered in any order, or evensimultaneously. If simultaneously, the multiple therapeutic agents areoptionally provided in a single, unified form, or in multiple forms (byway of example only, either as a single pill or as two separate pills).In some embodiments, one of the therapeutic agents is given in multipledoses, or both are given as multiple doses. If not simultaneous, thetiming between the multiple doses optionally varies from more than zeroweeks to less than four weeks. In addition, the combination methods,compositions and formulations are not to be limited to the use of onlytwo agents; the use of multiple therapeutic combinations is alsoenvisioned.

It is understood that the dosage regimen to treat, prevent, orameliorate the condition(s) for which relief is sought, is optionallymodified in accordance with a variety of factors. These factors includethe disorder from which the subject suffers, as well as the age, weight,sex, diet, and medical condition of the subject. Thus, the dosageregimen actually employed varies widely, in some embodiments, andtherefore deviates from the dosage regimens set forth herein.

The pharmaceutical agents which make up the combination therapydisclosed herein are optionally a combined dosage form or in separatedosage forms intended for substantially simultaneous administration. Thepharmaceutical agents that make up the combination therapy areoptionally also be administered sequentially, with either therapeuticagent being administered by a regimen calling for two-stepadministration. The two-step administration regimen optionally calls forsequential administration of the active agents or spaced-apartadministration of the separate active agents. The time period betweenthe multiple administration steps ranges from, a few minutes to severalhours, depending upon the properties of each pharmaceutical agent, suchas potency, solubility, bioavailability, plasma half-life and kineticprofile of the pharmaceutical agent. Circadian variation of the targetmolecule concentrations are optionally used to determine the optimaldose interval.

In addition, an antibody is optionally used in combination withprocedures that provide additional or synergistic benefit to thepatient. By way of example only, patients are expected to findtherapeutic and/or prophylactic benefit in the methods described herein,wherein pharmaceutical compositions of an antibody and/or combinationswith other therapeutics are combined with genetic testing to determinewhether that individual is a carrier of a mutant gene that is correlatedwith certain diseases or conditions.

An antibody and the additional therapy(ies) are optionally administeredbefore, during or after the occurrence of a disease or condition, andthe timing of administering the composition containing an antibodyvaries in some embodiments. Thus, for example, an antibody is used as aprophylactic and is administered continuously to subjects with apropensity to develop conditions or diseases in order to prevent theoccurrence of the disease or condition. An antibody and compositions areoptionally administered to a subject during or as soon as possible afterthe onset of the symptoms. The administration of the agents areoptionally initiated within the first 48 hours of the onset of thesymptoms, preferably within the first 48 hours of the onset of thesymptoms, more preferably within the first 6 hours of the onset of thesymptoms, and most preferably within 3 hours of the onset of thesymptoms. The initial administration is optionally via any routepractical, such as, for example, an intravenous injection, a bolusinjection, infusion over 5 minutes to about 5 hours, a pill, a capsule,transdermal patch, buccal delivery, and the like, or combinationthereof. An antibody is preferably administered as soon as ispracticable after the onset of a disease or condition is detected orsuspected, and for a length of time necessary for the treatment of thedisease, such as, for example, from about 1 month to about 3 months. Thelength of treatment optionally varies for each subject, and the lengthis then determined using the known criteria. For example, an antibody ora formulation containing an antibody are administered for at least 2weeks, preferably about 1 month to about 5 years, and more preferablyfrom about 1 month to about 3 years.

While certain embodiments, of the present invention have been shown anddescribed herein, it will be apparent to those skilled in the art thatsuch embodiments, are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat in some embodiments, of the invention various alternatives to theembodiments, described herein are employed in practicing the invention.

EXAMPLES Example 1

The following example demonstrates the production of B-cell linesderived from MMTV-tTA/TRE-MYC mice.

MMTV-tTA/TRE-MYC mice are maintained on doxycycline for eight weeksafter birth and then are switched to a normal diet. The mice develop anexternally evident lymphadenopathy and splenomegaly, and present with anumber of the clinical signs that are consistently seen associated withlymphoid neoplasia (scruffy fur, hunched posture, labored breathing,anemia, organomegaly, etc.). The mice are euthanized and their lymphnodes and spleens are collected for analysis. Single cell suspensionsare generated from some of the lymph nodes and a portion of the spleen.Those cells are used for flow cytometric analyses. The initialcharacterization of the tumors demonstrated the high prevalence ofactivated B-cells. Some of the same cells are used to seed cultures togenerate B-cell lines. These cells are cultured in lymphocyte media(RPMI 1640, 10% Fetal Calf Serum, penicillin/streptomycin, L-glutamine,HEPES, non-essential amino acids, sodium pyruvate and2-(3-mercaptoethanol). Approximately 14-21 days later, some of the wellsbegan to exhibit clonal outgrowth of cell lines. The cells are carefullyexpanded until they were adapted to growth in large flasks. A portion ofthe cells are cryopreserved in 10% DMSO.

Two cell lines were initially picked, designated TBLK6 and TBLK7. Thesecell lines exhibited different surface expression levels of CD138. Thelevels of immunoglobulin secretion into the tissue culture medium ismeasured after seeding. Both cell lines spontaneously secretedimmunoglobulin into the growth medium. The levels of secretion areincreased by the addition of IL-4 and IL-6 into the initial innoculumImmunoglobulins secreted by both TBLK6 and TBLK7 are shown to be IgM. Insome embodiments, single cells are cloned from both cell lines in orderto generate true monoclonal populations. A number of MMTV-tTA/TRE-MYCbigenic mice are generated that, in some embodiments, are used forisolating the AN1/T3 population by cell sorting.

Example 2

The following example demonstrates that antibodies produced inMyc-over-expressing mice function in vivo.

A model of lethal viral infection is used. Porcine Rabies Virus (PRV) isa member of the alpha-herpes viruses that has been previously shown tobe lethal in mice following intravenous administration. Two variants ofPRV are constructed. In one instance, one of the sequences for a genecalled US-9 was fused to GFP. This construct allowed for tracking of thevirus and virally infected cells in a different setting. US-9 proteinexpression is confirmed and US-9 retained its function as the fusionprotein. The virus is fully pathogenic in spite of its additionalgenetic cargo. A variant of PRV that encodes the open reading frame forHEL is also generated. This viral variant is shown to express HEL byWestern blot analysis of infected cells.

GFP-expressing virus or the HEL-expressing virus innocula are incubatedwith HEL-specific antibodies diluted 1:500 for one hour, on ice. Themixtures are then injected intravenously into groups of four mice. Themice are then monitored for four days following administration of thevirus and antibody mixtures.

As shown in FIG. 7, the GFP-expressing virus is not affected by thepresence of HEL-specific antibodies. The kinetics of mortality in thatgroup is comparable with our previous experience with wild type PRVstrains. In contrast, the kinetics of mortality in mice that receivedthe HEL-expressing virus is significantly delayed and that group of micelived for almost twice as long as the mice injected with theGFP-expressing virus. The viruses used for these experiments onlyexpressed the US-9 fusion protein transiently. After entry into cells,they produce wild type PRV.

Example 3

In this example, Myc-ER is used for the conditional immortalization oflong term hematopoietic stem cells. A group of bone marrow chimeric miceare generated using 5FU enriched bone marrow derived stem cells, asfollows. For bone marrow derived hematopoietic stem cells, 5 mg/mouse of5-fluorouracil (5FU) is administered intravenously, in order to enrichfor long-term hematopoietic stem cells, and induce HSC proliferation invivo. The bone marrow cells are collected from the femurs and tibiabones 5 days later. The red blood cells are lysed, using a hypotoniclysis buffer. The remaining cells are washed twice in media and platedat a concentration of 2×10⁶ cells/ml, in a 24 well plate, in DMEM mediasupplemented with 15% heat inactivated fetal calf serum,penicillin/streptomycin, L-glutamine, Non-essential amino acids,recombinant human IL-3, IL-6 and Stem Cell Factor (SCF). The cells arecultured for 24 hours prior to the first spin infection, and are subjectto the infection procedure 3 times, every 24 hours. A day after the lastspin infection, the cells are analyzed by flow cytometry. Lentivirallytransduced bone marrow derived hematopoietic stem cells arereconstituted to lethally irradiated mice, and are checked for GFPexpression in lymphoid organs 12 weeks later. In this instance,hematopoietic stem cells are allowed to reconstitute a normal peripherallymphoid compartment for 8-12 weeks after bone marrow transplantation.The splenic, GFP+ AN-1/T3 cells are isolated from those mice and areused for in vitro immortalization protocols, as described above. The keydifference is that instead of withdrawing doxycycline from the system,10 nM 4-hydroxytamoxifen (4OHT) is added to the medium. In someembodiments, sorted, GFP+ AN-1/T3 cells are adoptively transferred intocohorts of wild type recipient mice that are treated once weekly with 1mg/mouse of 4OHT, intraperitoneally. The mice are monitored daily forthe appearance of clinical signs associated with the development ofB-cell lymphomas. The resulting tumors are collected and used forgenerating B-cell lines as described herein, using 4OHT instead ofdoxycycline as the regulator of Myc function.

Example 4

In this example, Myc-ER and Bcl-2 are used for the conditionalimmortalization of long term hematopoietic stem cells. A group of bonemarrow chimeric mice are generated using 5FU enriched bone marrowderived stem cells, as follows. For bone marrow derived hematopoieticstem cells, 5 mg/mouse of 5-fluorouracil (5FU) is administeredintravenously, in order to enrich for long-term hematopoietic stemcells, and induce HSC proliferation in vivo. The bone marrow cells arecollected from the femurs and tibia bones 5 days later. The red bloodcells are lysed, using a hypotonic lysis buffer. The remaining cells arewashed twice in media and plated at a concentration of 2×10⁶ cells/ml,in a 24 well plate, in DMEM media supplemented with 15% heat inactivatedfetal calf serum, penicillin/streptomycin, L-glutamine, Non-essentialamino acids, recombinant human IL-3, IL-6 and Stem Cell Factor (SCF).The cells are cultured for 24 hours prior to the first spin infectionwith a GFP expressing lentivirus encoding the expression of Myc-ER andBcl-2. The cells are subject to the infection procedure 3 times, every24 hours. A day after the last spin infection, the cells are analyzed byflow cytometry. Lentivirally transduced bone marrow derivedhematopoietic stem cells are reconstituted to lethally irradiated mice.In this instance, hematopoietic stem cells are allowed to reconstitute anormal peripheral lymphoid compartment for 8-12 weeks after bone marrowtransplantation. The splenic, GFP+ AN-1/T3 cells are isolated from thosemice and are used for in vitro immortalization protocols, as describedabove. 4OHT is added to the medium to induce Myc activation. In someembodiments, sorted, GFP+ AN-1/T3 cells are adoptively transferred intocohorts of wild type recipient mice that are treated once weekly with 1mg/mouse of 4OHT, intraperitoneally. The mice are monitored daily forthe appearance of clinical signs associated with the development ofB-cell lymphomas. The resulting tumors are collected and used forgenerating B-cell lines as described herein, using 4OHT instead ofdoxycycline as the regulator of Myc function.

Example 5

In order to test the notion of harnessing the MMTV-tTA/TRE-MYC mice togenerate novel antibodies to antigens of interest through theintroduction into the system as neo-self antigens, a retroviral bonemarrow chimeric mice was used. A variant of pMIG that encodes the cDNAfor H5 hemagglutinin from a highly pathogenic avian influenza virus,A/Ty/Ont/7732/66 (H5N9) was first generated. HSCs enriched from the bonemarrows of mice that had been treated with 5-fluorouracil 5 days priorto collection, were then obtained as previously described. The donorcells were obtained from TRE-MYC mice. The cells were transduced withone retrovirus that encodes the tetracycline transactivator protein(tTA) as well as pMIG-H5. The transduced bone marrow HSCs were used toreconstitute lethally irradiated mice. The bone marrow chimeric micewere maintained on SEPTRA and observed daily for externally evidentclinical signs of hematological malignancies (scruffy fur, laboredbreathing, externally evident lymphadenopathy or splenomegaly, hunchedposture, dehydration, and hind limb paralysis). A cohort of 5 micepresented with clinical signs of hematological malignancies in a 7-dayperiod, starting 6 weeks after transplantation. The lymph nodes andspleens were collected from euthanized mice and used to generate singlecell suspensions. The remaining cells were either cultured to begin togenerate cell lines, or cryopreserved for subsequent analysis. A smallfraction of the cells were stained for cell surface markers and analyzedby flow cytometry (FIG. 8). FIG. 8 shows the surface profile of thecells isolated from the bone marrow chimeric mice that had developed ahematological malignancy. These are uniformly B220+/IgM+, suggestingthat the mice had developed a B-cell leukemia/lymphoma composed ofmature B-cells. These cells were placed in culture and clonally expandedpopulations were passed starting 8 days after initial seeding. This is asignificantly faster timeline than what is normally achieved withcurrent approaches to monoclonal antibody production. In addition, theaccelerated time frame by which this novel approach has allowed us togenerate novel antibodies to hemagglutinin should render this approachas a rapid response platform for the development of novel neutralizingantibodies to new and emerging infectious diseases and other biologicalthreats.

The serum from these mice was collected to test for the presence ofantibodies to H5 Immunoblot analysis with the serum from these miceshowed specific reactivity to the HA1 subunit of the H5 HA (FIG. 9).Cell lysates were obtained from 293FT cells that had been transientlytransfected with two different expression plasmids encoding H5(pCDNA3-H5 and pMIG-H5). The blots were probed with sera obtained fromleukemic mice. The banding pattern that developed was consistent with H5and the cleavage products that develop during the normal maturation andprocessing of HA in a cell. The mature HA is composed of two subunitsthe HA1 and the HA2. The HA1 subunit (˜40 kDa) forms the globular headof the molecule and is responsible for binding to the host cell sialicacid receptors on surface glycoproteins and glycolipids.

The HA2 (˜20 kDa) subunit is responsible for fusion of the viralenvelope with the endosomal membranes of the host cell during entry. Aprotease cleavage site separates these two subunits and cleavage by hostprotease is required for entry into the host cell. The vast majority ofvirus neutralizing epitopes are found in the HA1 subunit 22 and inhibitvirus-receptor interaction.

Heamagglutination inhibition analysis of mouse serum obtained fromretroviral chimeric mice was performed to test the ability of the serato block virus-receptor interaction with a variety influenza A isolatesincluding H5N2, H1N1, H7N2, H3N2, and H6N8 subtypes (FIG. 10).

Example 6

Mice (n=20) are divided into two groups. The first group (n=10) areimmunized with HEL mixed with Freund's adjuvant. The second group (n=10)are immunized with HEL mixed with Tat-Myc.

Both groups receive a booster shot of the HEL antigen mixed withIncomplete Freund's Adjuvant.

Sera is collected from each mouse at days 4, 7, 14, 21 and 28.Reactivity to HEL protein is assayed by ELISA.

On day 35, spleens are harvested from each mouse. The spleens areprocessed into single cell suspension. Red blood cells (RBCs) areremoved with lysis buffer. Lymphocytes are divided into two samples. Thefirst sample is incubated with Tat-Myc and HEL antigen. The secondsample is incubated with TAT-MYC.

Example 7

Mice (n=20) are divided into two groups. The first group (n=10) areimmunized with Fluvirin (2007-2008) mixed with Freund's adjuvant. Thesecond group (n=10) are immunized with Fluvirin (2007-2008) mixed withTat-Myc.

Both groups receive a booster shot of the Fluvirin (2007-2008) mixedwith Incomplete Freund's Adjuvant (IFA).

Sera is collected from each mouse at days 4, 7, 14, 21 and 28.Reactivity to Fluvirin (2007-2008) is assayed by ELISA.

On day 35, spleens are harvested from each mouse. The spleens areprocessed into single cell suspension. Red blood cells (RBCs) areremoved with lysis buffer. Lymphocytes are divided into two samples. Thefirst sample is incubated with Tat-Myc and Fluvirin (2007-2008). Thesecond sample is incubated with TAT-MYC.

It should be understood that various alternatives to the embodiments, ofthe invention described herein may be employed in practicing theinvention. It is intended that the following claims define the scope ofthe invention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1.-20. (canceled)
 21. A fusion peptide, comprising: (a) a proteintransduction domain sequence; (b) a MYC sequence having a biologicalactivity of MYC; and (c) at least two protein tags; wherein the fusionpeptide is able to enter the nucleus of a cell.
 22. The fusion peptideof claim 21, wherein the protein transduction domain sequence is an HIVTat protein transduction domain sequence.
 23. The fusion peptide ofclaim 22, wherein the Tat protein transduction domain sequence isselected from the group consisting of TAT_([48-57]) and TAT_([57-48]).24. The fusion peptide of claim 22, wherein the Tat protein transductiondomain sequence is SEQ ID NO:6.
 25. The fusion peptide of claim 21,wherein the protein transduction domain sequence is a vpr proteintransduction domain sequence.
 26. The fusion peptide of claim 21,wherein the two protein tags comprise one or more of a polyhistidine tagor an epitope tag.
 27. The fusion peptide of claim 21, wherein the twoprotein tags are a 6-histidine tag and a V5 epitope tag.
 28. The fusionpeptide of claim 21, wherein the MYC sequence is SEQ ID NO:1.
 29. Thefusion peptide of claim 21, wherein the fusion peptide comprises SEQ IDNO:5.
 30. The fusion peptide of claim 21, wherein the fusion peptide hasthe following sequence:MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH.


31. The fusion protein of claim 21, wherein the MYC activity comprisesregulating transcriptional activity of Myc responsive genes.
 32. Thefusion protein of claim 21, wherein the MYC activity comprises antibodyproduction.
 33. The fusion protein of claim 21, wherein the MYC activitycomprises cell proliferation.
 34. A composition comprising: (a) a fusionpeptide, comprising: (i) a protein transduction domain sequence; (ii) aMYC sequence having a biological activity of MYC; and (iii) at least twoprotein tags; wherein the fusion peptide is able to enter the nucleus ofa cell; and (b) one or more pharmaceutically acceptable excipients. 35.The composition of claim 34, wherein the protein transduction domainsequence is an HIV Tat protein transduction domain sequence.
 36. Thecomposition of claim 35, wherein the Tat protein transduction domainsequence is selected from the group consisting of TAT_([48-57]) andTAT[_(57-48]).
 37. The composition of claim 35, wherein the Tat proteintransduction domain sequence is SEQ ID NO:6.
 38. The composition ofclaim 34, wherein the protein transduction domain sequence is a vprprotein transduction domain sequence.
 39. The composition of claim 34,wherein the two protein tags comprise one or more of a polyhistidine tagor an epitope tag.
 40. The composition of claim 34, wherein the twoprotein tags are a 6-histidine tag and a V5 epitope tag.
 41. Thecomposition of claim 34, wherein the MYC sequence is SEQ ID NO:
 1. 42.The pharmaceutical composition of claim 31, wherein the fusion peptidecomprises SEQ ID NO:5.
 43. The composition of claim 34, wherein thefusion peptide has the following sequence:MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH.


44. The composition of claim 34, wherein the MYC activity comprisesregulating transcriptional activity of Myc responsive genes.
 45. Thecomposition of claim 34, wherein the MYC activity comprises antibodyproduction.
 46. The composition of claim 34, wherein the MYC activitycomprises cell proliferation.